Media supplements and methods to culture human gastrointestinal anaerobic microorganisms

ABSTRACT

A media supplement for culturing anaerobic bacteria is provided which comprises a filtrate of eilluent from a chemostat vessel in which a target bacterial ecosystem has been culnn-ed. Methods of using the supplement for culturing or isolating anaerobic microbial strains or cormmmities, particularly anaerobic bacteria from the human gut, are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/433,364, filed Jun. 6, 2019, which is a continuation of U.S.application Ser. No. 15/440,906, filed on Feb. 23, 2017, now U.S. Pat.No. 10,314,864, which is a continuation of U.S. application Ser. No.14/344,967, filed on May 5, 2014, which is a 371 national phaseapplication off of International Application No. PCT/CA2012/050641,filed Sep. 14, 2012, which claims priority to U.S. ProvisionalApplication No. 61/534,456, filed on Sep. 14, 2011, the entire contentsof which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to methods for culturing microbial communitiesfrom the human distal colon and methods for isolating anaerobic microbesfrom such communities, as well as media supplements for use in suchmethods.

BACKGROUND OF THE INVENTION

The human gut is the most densely inhabited ecosystem on Earth (Marchesiand Shanahan, 2007). Like other complex microbial ecosystems, the humanmicrobiota has not been sampled to completion (Eckburg et al., 2005).This is because the individual species of the gut microbiota aredifficult to culture axenically in vitro (Hart et al., 2002). In fact,of the 500+ bacterial species which colonize the human intestinal tract,about 75% have not been cultured using conventional techniques (Duncanet al., 2007; Eckburg et al., 2005; Hayashi et al., 2002). It isrecognized that novel culture techniques are required to grow these“unculturable” microorganisms.

Studies of gut microbiota have been hampered by a lack of model systems.While in vivo models can provide researchers with physiologicallyrelevant experimental models, they have several drawbacks. For example,different study participants can each have unique, host-specificcommunity profiles representing their gut microbiota, making comparisonof the gut microbiota between subjects difficult, especially whenattempting to correlate the effects of a treatment to changes in the gutmicrobiota. In vivo models also often limit the dynamic monitoring ofthe gut microbiota by deriving their data from end-point measurements.Experiments involving humans or animals require research ethics approvalwhich can limit the experiments conducted on an individual's gutmicrobiota in vivo.

In an attempt to improve upon the drawbacks of in vivo models, severalin vitro models have been developed. These in vitro systems range fromsimple batch culture vessels to complex continuous culture or‘chemostat’ systems (Macfarlane, G. T. and Macfarlane, S., Curr. Opin.Biotechnol., 18(2): 156-62, 2007). Using chemostats, communities seededfrom fresh feces can reach a steady-state that closely resembles in vivodistal gut communities. Being a host-free system, chemostats supportinggut microbiota make ideal vessels in which to study microbialperturbations that directly result from the addition of exogenousstimuli in isolation from the effects of these stimuli on hostphysiology, making them useful for mechanistic studies (Macfarlane, G.T. and Macfarlane, S., Curr. Opin. Biotechnol., 18(2): 156-62, 2007).

In vitro models also provide several other advantages over in vivomodels in studies of the human gut microbiota. In vitro studies aregenerally inexpensive and easy to set-up. They also allow for the strictcontrol of factors that influence the environment while stillfacilitating frequent and simple sampling of the simulated gutcommunities. However, while chemostats provide a useful tool toinvestigate the microbial ecology of the gut, operational parametersvary widely between different models in different laboratories, oftenwithout experimental validation. Preparation of the inocula, compositionof the media, and retention time of the vessel are parameters which canvary between different studies.

To represent a valid model of the human distal gut, communities whichdevelop within chemostat vessels should share some similarity to thefecal inoculum from which the gut community was derived. The microbialecosystem of the gut is a highly diverse community, and it is thereforeimportant that communities grown in artificial systems also retain ahigh level of diversity (including species richness and evenness).Finally, the reproducibility and stability of these communities must beestablished and characterized before experimentation can begin. Thismeans that microbial communities developed within these models must bethoroughly analyzed and compared to in vivo communities before thevalidity of a system can be confirmed.

Chemostat and fecal communities can be monitored using molecular methodssuch as Denaturing Gradient Gel Electrophoresis (DGGE). Currently, thereis a lack of standardization between DGGE analysis methods used indifferent research laboratories. Methods of DGGE analysis vary fromvisual inspection to methods utilizing statistical analysis software(such as GelCompar™, BioNumerics, GeneTools, Quantity One™, etc.).Monitoring of communities using computer software allows for morereliable and detailed analysis of DGGE gels and provides more data onthe composition and structure of microbial communities, the stability ofthe community, and the similarity between profiles. However,laboratories utilizing these analysis programs do not use consistentmethods when analyzing their DGGE gels and report varying data on theircommunities. If the analysis of DGGE gels can be standardized then thiswill facilitate the comparison between the chemostat communities fromdifferent laboratories.

It would be desirable therefore to be provided with chemostat models ofthe human distal gut that are stable, reproducible and biologicallysignificant, as well as more complete methods for the assessment andverification of such models.

SUMMARY OF THE INVENTION

There are provided herein methods for culturing microorganisms thatnormally live in the human large intestine. Methods for developing andcharacterizing microbial communities from the human distal colon and forassessing and/or verifying such communities are provided herein.

In an aspect, there are provided methods for culturing microorganismsfrom the human distal colon using a media supplement termed “LiquidGold”. Liquid Gold refers to a 0.2 μm filtrate of spent culture media oreffluent from a chemostat vessel in which a target ecosystem iscultured. Liquid Gold is used to supplement culture media, e.g.,standard laboratory media. Supplementation with Liquid Gold allowsculturing of “unculturable” microorganisms, i.e., microorganisms whichare refractory to culture axenically using traditional methods. Withoutwishing to be limited by theory, it is believed that Liquid Goldprovides ‘growth signals’ to previously uncultured microbes to enhancetheir axenic growth in vitro and hence allow them to be cultured andgrown in vitro. It is known in the art that certain microbes may growwell within a microbial ecosystem, but are refractory to growth inisolation; presumably the larger bacterial community in the ecosystem insome way “supports” the growth of the microbes. We report herein thatthis support can be provided by Liquid Gold to allow isolation ofcertain microbes and to establish their growth as a pure isolate invitro, separate from the rest of the ecosystem.

Thus, in an aspect there is provided a method for growing anaerobicbacteria comprising culturing the bacteria in a chemostat underconditions replicating normal human colonic gastrointestinal tract inequilibrium and then purifying individual anaerobic bacteria into pureisolates.

In another aspect, there are provided microbial communities from thehuman distal colon. In an embodiment, there is provided a single-stagechemostat model of the human distal gut. In an embodiment, microbialcommunities are stable, reproducible, and/or biologically significant.

In another aspect, there is provided herein a media supplement forculturing microbes termed “Liquid Gold.” “Liquid Gold” refers tofiltered effluent from the chemostat, i.e., the effluent forced out ofthe chemostat through pressure differentials; it drips into sterilebottles, housed behind the chemostat, via tubing. When the bottle isfull, it is sealed and can be stored at +4° C. until needed. Theeffluent is passed through a 0.2 μm, e.g, a 0.22 μm filter (Durapore,Millipore, USA), to remove bacterial cells to produce cell-free LiquidGold, which is used to supplement culture media (usually added to 1%v/v, 3% v/v, 5% v/v, 7% v/v or 10% v/v). Liquid Gold is essentiallysupernatant from a culture of microbes, containing a plethora ofsignaling molecules, growth factors and so on. In an embodiment, LiquidGold is used to supplement culture media at 3% v/v. It should beunderstood that Liquid Gold will differ depending on the microbialcommunity from which it is produced.

In an embodiment, there is provided a media supplement for culturinganaerobic bacteria, the media supplement comprising a filtrate ofeffluent from a chemostat vessel in which a target bacterial ecosystemhas been cultured. The filtrate may be, e.g., a 0.2 μm filtrate. In oneembodiment, the culture media in which the target bacterial ecosystem iscultured is standard culture media. In another embodiment, the culturemedia is Media 1. In an embodiment, the culture media comprises mucin.Mucin may be present in the culture media at a concentration of about1-10%. In an embodiment, mucin is present in the culture media at aconcentration of 4 g/L. In an embodiment, a human fecal sample has beencultured in the chemostat. The human fecal sample may be, for example, a10% w/v fecal slurry supernatant or a 20% w/v fecal slurry supernatant.In another embodiment, Defined Experimental Community 1 (DEC-1), DefinedExperimental Community 2 (DEC-2) or Defined Experimental Community 3(DEC-3) has been cultured in the chemostat. In yet another embodiment,the target bacterial ecosystem comprises at least one, at least three,at least five, at least 8, at least 10, at least 15, or at least 25 ofthe bacterial strains listed in Table 1, Table 2 or Table 3. In afurther embodiment, the target ecosystem which has been cultured in thechemostat comprises a community of bacterial strains representing anenterotype of human gut, e.g., the Bacteroides, the Prevotella or theRuminococcus enterotype. In another embodiment, the anaerobic bacteriaare bacteria found in the human gut microbiome.

In an embodiment, the chemostat vessel used in the methods andpreparations of the invention is a single-stage chemostat.

In an embodiment, there is provided a media supplement for culturinganaerobic bacteria, wherein the media supplement is prepared by: a)culturing a target bacterial ecosystem in culture media in asingle-stage chemostat under conditions replicating normal human colonicgastrointestinal tract, in equilibrium; b) collecting effluent from thechemostat; and c) filtering the effluent through a 0.2 μm filter toremove bacterial cells, in order to produce the media supplement. In anembodiment, the method for preparing the media supplement furthercomprises a step of centrifuging the effluent at 14,000 rpm for 10minutes and collecting the supernatant before step c), wherein theeffluent supernatant is then filtered in step c). In another embodiment,the method for preparing the media supplement further comprisesfiltering the effluent or effluent supernatant sequentially through a1.0 μm filter, a 0.8 μm filter, and a 0.45 μm filter, before filteringthrough the 0.2 μm filter. In an embodiment, the 0.2 μm filter is a 0.22μm filter. The culture media may be, e.g., standard culture media orMedia 1.

In an embodiment, the target bacterial ecosystem is obtained byculturing a human fecal sample, e.g., a 10% w/v fecal slurry supernatantor a 20% w/v fecal slurry supernatant. In another embodiment, the targetbacterial ecosystem comprises Defined Experimental Community 1 (DEC-1),Defined Experimental Community 2 (DEC-2), or Defined ExperimentalCommunity 3 (DEC-3). In another embodiment, the target bacterialecosystem comprises a community of bacterial strains representing anenterotype of human gut, e.g., the Bacteroides, the Prevotella or theRuminococcus enterotype. In yet another embodiment, the target bacterialecosystem comprises at least one, at least three, at least five, atleast 8, at least 10, at least 15, or at least 25 of the bacterialstrains listed in Table 1, Table 2 or Table 3.

In an embodiment, the culture media is Media 1. In another embodiment,the culture media comprises mucin, e.g., at a concentration of 1-10%,e.g., at a concentration of 4 g/L. In an embodiment, the chemostat is asingle-stage chemostat. In an embodiment, the chemostat has a systemretention time of 24 hours. In another embodiment, the conditionsreplicating normal human colonic gastrointestinal tract comprise: atemperature of about 37° C.; a pH of about 6.9 to 7; a system retentiontime of 24 hours; and maintenance of anaerobic conditions in thechemostat. In another embodiment, the conditions replicating normalhuman colonic gastrointestinal tract further comprise culturing thetarget bacterial ecosystem in culture media containing mucin, e.g.,mucin at a concentration of 1-10%, e.g., 4 g/L.

In an embodiment, there is provided herein a use of the media supplementof the invention for growing anaerobic bacteria, wherein the mediasupplement is used to supplement culture media in a liquid culture atabout 1% v/v to about 10% v/v. In an embodiment, the media supplement isused to supplement culture media at about 3% v/v. In an embodiment, theliquid culture is grown in a chemostat. The media supplement may beadded to the culture media before culturing begins, or during culturingof the anaerobic bacteria. There is also provided herein a use of themedia supplement of the invention for growing anaerobic bacteria,wherein the media supplement is used to supplement solid culture media,e.g., solid culture media in a Petri dish. In an embodiment, the mediasupplement is added to FAA plates at a final concentration of 3%.

In some embodiments, for the uses provided herein, the anaerobicbacteria are bacteria found in human gut of a healthy subject. In anembodiment, the anaerobic bacteria are Faecalibacterium prausnitzii orRuminococcus callidus (ATCC27760).

In an embodiment, there is provided herein a method of isolatinganaerobic bacteria from human gut, comprising: a) culturing a targetbacterial ecosystem in culture media (e.g., standard culture media,Media 1) in a single-stage chemostat under conditions replicating normalhuman colonic gastrointestinal tract, until equilibrium is reached; b)diluting the culture and plating onto Fastidious anaerobe agar (FAA)supplemented with the media supplement of the invention, and optionallysupplemented with defibrinated sheep blood; c) incubating plates in ananaerobe chamber; d) purifying individual anaerobic bacterial coloniesgrown in step (c); and e) optionally, culturing the purified individualanaerobic bacterial colonies from step (d) in liquid culture in asingle-stage chemostat under conditions replicating normal human colonicgastrointestinal tract, optionally wherein the media supplement of theinvention is used to supplement culture media at about 1% v/v to about10% v/v; such that isolates of anaerobic bacteria are obtained. In anembodiment, the media supplement is used to supplement the culture mediain step (e) at about 3% v/v. In another embodiment, the media supplementis added at a final concentration of 3% in step (b). In yet anotherembodiment, the defibrinated sheep blood is added at a finalconcentration of 5%. In a still further embodiment, the conditionsreplicating normal human colonic gastrointestinal tract comprise: atemperature of about 37° C.; a pH of about 6.9 to 7; a system retentiontime of 24 hours; and maintenance of anaerobic conditions in thechemostat. In an embodiment, the conditions replication normal humancolonic gastrointestinal tract further comprise culturing in culturemedia to which mucin has been added. In an embodiment, bacteria arecultured in the chemostat in steps (a) and (e) under reduced atmospherewith controlled levels of partial pressure of N₂:CO₂:H₂. For example,the preparation may be under N₂, CO₂ or H₂, or a mixture thereof. In anembodiment, the mixture thereof is N₂:CO₂:H₂. In another embodiment,anaerobic conditions are maintained by bubbling filtered nitrogen gasthrough the cultures in steps (a) and (e).

In some embodiments, the target bacterial ecosystem cultured in step (a)is a human fecal sample, e.g., a 10% w/v fecal slurry supernatant or a20% w/v fecal slurry supernatant. In an embodiment, standard culturemedia is used in the chemostat. In an embodiment, Media 1 is used in thechemostat.

In an embodiment, Faecalibacterium prausnitzii or Ruminococcus callidus(ATCC27760) is isolated. In another embodiment, a pure isolate ofFaecalibacterium prausnitzii, Clostridium aldenense 1, Clostridiumaldenense 2, Clostridium hathewayi 1, Clostridium hathewayi 2,Clostridium hathewayi 3, Clostridium thermocellum, Ruminococcus bromii2, Ruminococcus torques 4, Ruminococcus torques 5, Clostridium cocleatum(e.g., Clostridium cocleatum 21 FAA1), Eubacterium desmolans (e.g.,Eubacterium desmolans 48FAA1), Eubacterium limosum 13LG, Lachnospirapectinoshiza, Ruminococcus productus (e.g., Ruminococcus productus27FM), Ruminococcus obeum (e.g., Ruminococcus obeum 11FM1), Blautiaproducta, or Clostridium thermocellum is obtained.

In an embodiment, there is provided herein a pure isolate ofFaecalibacterium prausnitzii, Clostridium aldenense 1, Clostridiumaldenense 2, Clostridium hathewayi 1, Clostridium hathewayi 2,Clostridium hathewayi 3, Clostridium thermocellum, Ruminococcus bromii2, Ruminococcus torques 4, Ruminococcus torques 5, Clostridium cocleatum(e.g., Clostridium cocleatum 21 FAA1), Eubacterium desmolans (e.g.,Eubacterium desmolans 48FAA1), Eubacterium limosum 13LG, Lachnospirapedinoshiza, Ruminococcus productus (e.g., Ruminococcus productus 27FM),Ruminococcus obeum (e.g., Ruminococcus obeum 11FM1), Blautia producta,or Clostridium thermocellum. A pure isolate may be obtained, e.g., usingmethods provided herein.

In an embodiment, there is provided a method of culturing a microbialcommunity from human gut, comprising: a) obtaining a fecal sample from ahealthy human subject; b) inoculating a culture with the fecal sample;and c) culturing the culture in culture media (e.g., standard culturemedia, Media 1, etc.) in a single-stage chemostat under conditionsreplicating normal human colonic gastrointestinal tract, untilequilibrium is reached; such that a microbial community comprisingbacterial strains found in human gut is obtained. In an embodiment, themicrobial community represents a human gut enterotype. In anotherembodiment, the fecal sample obtained in step (a) is prepared as a 10%w/v fecal slurry supernatant or a 20% w/v fecal slurry supernatantbefore inoculating the culture in step (b).

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

For a better understanding of the invention and to show more clearly howit may be carried into effect, reference will now be made by way ofexample to the accompanying drawings, which illustrate aspects andfeatures according to preferred embodiments of the present invention,and in which:

FIG. 1 shows a single-stage chemostat vessel developed by modifying aMultifors fermentation system which was used for growing the isolatedbacterial strains as described herein.

FIG. 2 shows a clustering Tree based on Dice similarity coefficient andUnweighted Pair Group Method with Arithmetic Mean (UPGMA) correlation ofthe DGGE profiles showing the 10% fecal inocula prepared from Donor 2feces on several different donations over an 8 month period. Thepredominant bacterial species from this healthy donor remained stableover time.

FIG. 3 shows a clustering Tree based on Dice similarity coefficient andUPGMA correlation of the DGGE profiles showing the 10% fecal inoculaprepared from four different donors (donors 1-4). Each donor had aunique profile, with the profiles from some donors more similar to eachother than others (e.g., Donors 2 and 3).

FIG. 4 shows a clustering Tree based on Dice similarity coefficient andUPGMA correlation of the DGGE profiles showing the 10% vs. 20% fecalinocula prepared from Donor 2 feces on two different donations. The 10%and 20% inocula were very similar to each other, therefore justifyingthe use of the 10% inocula (which requires less fecal donation and iseasier to administer to the chemostat vessel upon inoculation).

FIGS. 5A-F shows the reproducibility of two chemostat vessels (V1 andV2) seeded with identical fecal inoculum from Donor 2. A) DGGE profilesshowing communities on days 0, 10, 26 and 28; B) Correlationcoefficients (expressed as percentages) comparing the profiles of eachvessel at the same time point, plotted over the course of theexperiment; C) Community dynamics as shown using moving windowcorrelation analysis. Similarity of the community within each vessel wascalculated by comparing the profile of day (x) and day (x-2); D) ShannonDiversity Index (H′) plot representing the community diversity of eachvessel over the course of the experiment; E) Range weighted richness(Rr) plot representing the richness in each vessel over the course ofthe experiment; F) Shannon equitability index (EH) plot representing thecommunity evenness values from each vessel over the course of theexperiment. Without mucin, two vessels could be run in parallel andmaintain identical communities, reaching steady state at about 26-28days post-inoculation.

FIGS. 6A-F show a comparison of the chemostat media used in ourlaboratory (“Medial”; used to feed V1) to a previously published medium(V6; Walker et al., Appl. Environ. Microbiol., 71 (7):3692-700, 2005).The same fecal inoculum (from Donor 2, 10%) was used to seed eachvessel. A) DGGE profiles showing communities on days 0, 10, 26 and 36;B) Correlation coefficients (expressed as percentages) comparing theprofiles of each vessel at the same time point, plotted over the courseof the experiment; C) Community dynamics as shown using moving windowcorrelation analysis. Similarity of the community within each vessel wascalculated by comparing the profile of day (x) and day (x-2); D) ShannonDiversity Index (H′) plot representing the community diversity of eachvessel over the course of the experiment; E) Range weighted richness(Rr) plot representing the richness in each vessel over the course ofthe experiment; F) Shannon equitability index (EH) plot representing thecommunity evenness values from each vessel over the course of theexperiment. Comparison shows that the media recipe we developed (Medial)provides a suitable medium to grow a stable and diverse chemostatcommunity when compared to the previously published medium.

FIGS. 7A-F show a comparison of a 65 hour retention time (V1) to a 24hour retention time (V2). The same fecal inoculum (from Donor 2, 10%)was used to seed each vessel. A) DGGE profiles showing communities ondays 0, 6, 10 and 14; B) Correlation coefficients (expressed aspercentages) comparing the profiles of each vessel at the same timepoint, plotted over the course of the experiment; C) Community dynamicsas shown using moving window correlation analysis. Similarity of thecommunity within each vessel was calculated by comparing the profile ofday (x) and day (x-2); D) Shannon Diversity Index (H′) plot representingthe community diversity of each vessel over the course of theexperiment; E) Range weighted richness (Rr) plot representing therichness in each vessel over the course of the experiment; F) Shannonequitability index (EH) plot representing the community evenness valuesfrom each vessel over the course of the experiment. Increasing theretention time from the biologically significant value of 24 hours to 65hours resulted in a community which was less similar to its inoculum anddid not maintain a higher level of diversity.

FIGS. 8A-F show the effect of mucin on the diversity of distal gutcommunities grown in a single-stage chemostat. The same fecal inoculum(from Donor 2, 10%) was used to seed each vessel. A) DGGE profilesshowing communities on days 0 and 24; B) Correlation coefficients(expressed as percentages) comparing the profiles of V1 (no mucin) to V5and V6 (with mucin) on days 0 and 24; C) Correlation coefficients(expressed as percentages) comparing the profiles of V1 (no mucin) tothe average values from V5 and V6 (with mucin) on days 0 and 24;D)Shannon Diversity Index (H′) representing the community diversity ofeach vessel on days 0 and 24; E) Range weighted richness (Rr)representing the richness in each vessel on days 0 and 24; F) Shannonequitability index (J) representing the community evenness values fromeach vessel on days 0 and 24. Addition of mucin to the chemostatresulted in increases in community diversity, richness, and evenness.

FIG. 9 shows a schematic description of measures used to characterizemicrobial ecological communities (dynamics, diversity, evenness andrichness). The schematic diagram explains basic ecological concepts(including community dynamics, diversity, evenness, and richness). A)Community dynamics represents the changes within the community over afixed time frame using moving window correlation analysis (Marzorati, M.et al., Environ. Microbiol., 10: 1571-1581, 2008; Possemiers, S. et al.,FEMS Microbiol. Ecol., 49: 495-507, 2004); B) Shannon diversity index isa measure of community diversity which takes both species richness(number of species present) and evenness (relative species abundance)into account (Gafan, G. P. et al., J. Clin. Microbiol., 43: 3971-3978,2005); C) Shannon equitability index describing community evenness, orthe degree to which the numbers of individuals are evenly dividedbetween the different species of the community (Pielou, E. C. 1975.Ecological diversity. Wiley, New York); D) Community richness refers tothe number of species present in the ecosystem; this measure does nottake relative species abundance into account.

FIG. 10 depicts DGGE profiles comparing fecal communities to thecommunities present in the chemostat vessels immediately followinginoculation. Two different chemostat runs were compared for each healthydonor (Donors 5 and 6). The fecal inocula used to seed the chemostatvessels was very similar to the starting fecal donation and not alteredsignificantly by the process of preparing the inoculum.

FIG. 11 depicts DGGE profiles comparing fecal communities from twodifferent healthy donors (Donors 5 and 6). Each donor provided a sampleon two different occasions. Donors 5 and 6 had different DGGE profiles.The DGGE profiles from both donors were consistent between donations.

FIG. 12 depicts DGGE profiles comparing fecal communities present in thechemostat vessels immediately following inoculation to the steady statecommunities (samples obtained 36 days post-inoculation) for twodifferent healthy donors (donors 5 and 6). Two different vessels wereseeded with identical fecal inoculum for each chemostat run and twodifferent chemostat runs were compared for each healthy donor. By DGGE,the fecal inocula from the same donor on two different occasions weremore similar to each other than to fecal inocula from the other donor.Also, the steady state communities seeded with feces from the same donorwere more similar to each other between chemostat runs than to thecommunities seeded with feces from another donor.

FIGS. 13A-F show community analysis of two identical chemostat vesselsmodeling the human distal gut. Each vessel was seeded with identicalfecal inocula prepared from the feces of a healthy donor (donor 5).Parameters were calculated by analyzing DGGE patterns of generalBacteria (V3 region of the 16S gene) using GeneTools statisticalanalysis software. Samples were analyzed every two days throughout theduration of the experiment (days 0-48). The vertical dashed linerepresents the beginning of steady state conditions. A: Correlationcoefficients (expressed as percentages) comparing the profiles of eachvessel at the same time point, plotted over the course of theexperiment. The horizontal dotted line represents the cut-off thresholdcalculated by comparing the similarity of identical marker lanes run ona single DGGE gel. The horizontal dashed line represents the cut-offthreshold −5% and allows for a 5% difference in similarity between theprofiles of each vessel. Up until day 48, both vessels were able tomaintain very similar DGGE profiles. B: Correlation coefficientscomparing the profiles of samples taken from each vessel over the courseof the experiment to its starting inocula. The horizontal dotted linerepresents the cut-off threshold and the horizontal dashed linerepresents the cut-off threshold −5%. While the steady state communitywas different from the starting inoculum, the similarity was relativelyconsistent over time. C: Community dynamics as shown using moving windowcorrelation analysis. Variability of the community within each vesselwas calculated by comparing the profile of day (x) and day (x-2). Thehorizontal dotted line represents 100-(cut-off threshold) and thehorizontal dashed line represents 100-(cut-off threshold −5%). By day 36the communities within both vessels had reached steady state (whenconfirmed by visual inspection of the DGGE profiles between vessels). D:Shannon Diversity Index (H) plot representing the corrected communitydiversity of each vessel over the course of the experiment. Thehorizontal dotted line represents the average Shannon diversity indexvalue of the starting inocula. The diversity in both vessels was similarto each other, stable over time, and similar to that of the startinginocula. E: Community richness (S) plot represented by plotting thenumber of corrected observed bands in each DGGE gel against time. Thehorizontal dotted line represents the average richness value of thestarting inocula. The richness in both vessels was similar to eachother, stable over time, and similar to that of the starting inocula. F:Shannon equitability index (EH) plot representing the correctedcommunity evenness values from each vessel over the course of theexperiment. The horizontal dotted line represents the average Shannonequitability index value of the starting inocula. The evenness in bothvessels was similar to each other, stable over time, and similar to thatof the starting inocula.

FIGS. 14A-B show representative plates demonstrating growth of theFaecalibacterium prausnitzii strain, which showed differential growth inresponse to Liquid Gold media supplement included in the agar mediapreparation at 3%. Plates were inoculated with identical inocula andincubated at 37° C. for 3 days under total anaerobic conditions. PlateA: Fastidious anaerobe agar (FAA) supplemented with 5% defibrinatedsheep blood alone. Plate B: FAA supplemented with 5% defibrinated sheepblood and 3% filtered (cell-free) Liquid Gold media supplement (fromDonor 6). Growth was clearly enhanced by addition of Liquid Gold mediasupplement to the media.

DETAILED DESCRIPTION OF THE INVENTION

According to a broad aspect of the invention there are provided hereinnovel methods of culturing anaerobic microorganisms or microbes using asingle-stage chemostat system. There is also provided a novel mediasupplement, termed “Liquid Gold,” for use in culturing suchmicroorganisms or microbes, in particular those which are traditionallydifficult to grow. Methods provided herein can be used, inter alia, toculture enterotypes of the human gut and to provide models of bacterialcommunities of the human distal colon. Methods provided herein areparticularly suited to culturing anerobic bacteria, such as those foundin the human gut.

The human gastrointestinal tract contains vast numbers of bacteria,collectively called the intestinal microbiota. The commensal gut floracontribute to host defense by priming the dendritic cells of the immunesystem, producing bactericidal products that kill pathogenic bacteria,inhibiting the colonization of pathogenic bacteria and competing withpathogens for food and for binding sites along the intestinal epithelialcell surface, a phenomenon collectively known as “colonizationresistance” (Stecher B. and Hardt W. D., Trends Microbiol. (2008),16:107-14; Rolfe, R. D., Infect. Immun. (1984), 45:185-91).

Recent studies have suggested that intestinal or gut enterotypes may notbe specific to an individual but, rather, are representative ofdifferent states of equilibrium that exist in the gut microbiota inresponse to dietary stimuli. It has been reported that the human gutmicrobiome, that is, the community of organisms that live symbioticallywithin humans, occurs in certain set varieties or “enterotypes. “Threemain enterotypes of the human gut, which vary in species and functionalcomposition, have been identified to date, and are termed Bacteroides,Prevotella and Ruminococcus (Arumugam, M. et al., Nature 12; 473(7346):174-80, Epub Apr. 20, 2011).

In an aspect, there are provided herein culture methods for culturingenterotypes of the human gut. As reported herein, we harvested fecalsamples from donors of each enterotype and used a single-stage chemostatsystem to culture a bacterial community modelling the community of thedonor's distal intestine or gut. Thus, in an embodiment there areprovided herein methods for culturing bacterial communities which modelenterotypes of the human gut. Methods provided herein can be used toform microbial communities which are stable, reproducible, diverse,and/or biologically significant, in terms of modeling the human distalcolon. As reported herein, steady-state communities are generallyproduces at about one-month, e.g., at approximately 26 days,post-inoculation with a fecal sample, using methods described herein.

In addition, there are provided herein three Defined ExperimentalCommunities (DECs) of microorganisms from human fecal samples. Asdescribed, using methods provided herein we subcultured fecal samplesfrom donors representing different enterotypes. Fecal samples wereharvested and used to generate DECs of microorganisms modelingenterotypes of the human gut. In an embodiment, three DECs (referred toherein as DEC-1, DEC-2 and DEC-3) are provided.

DEC-1 comprises intestinal bacterial strains that were isolated andpurified from donor stool from a donor who had not received antibioticsin the last 5 years (this donor is also referred to herein as “Donor6”). DEC-1 includes 33-strains (representing a total of 26 species), asshown in Table 1; this DEC has been used successfully to treat twopatients (Kingston General Hospital) with recurrent Clostridioides(Clostridium) difficile infection, demonstrating that the DECsuccessfully models a microbial community of the human distal colon.Strains were speciated using the 16S rRNA full-length sequence and theGreenGenes database accessible via Lawrence Berkley National Laboratory(1b1) website.

DEC-2 (from the same donor as DEC-1) includes all the strains in DEC-1as well as additional bacterial species. Bacterial strains included inDEC-2 are shown in Table 3.

DEC-3 includes isolates of bacterial species shown in Table 2. DEC-3 wasisolated from a male donor, 43 yrs old, with no history of antibioticuse in the 6 years prior to stool donation (referred to herein as “Donor5”). Notably, DEC-3 contains a number of microbes which are either knownor speculated to be highly beneficial and enriched in healthyindividuals, such as Akkermansia muciniphila, Faecalibacteriumprausnitzii, Bifidobacterium spp., and Adlercreutzia equolifasciens.

In an embodiment, methods are provided herein for culturing bacterialcommunities which model enterotypes of the human gut use a single-stagechemostat system. This system has been optimized for growinggastrointestinal microbes. In an embodiment, there is provided achemostat system using the following culture media, referred to hereinas “Media 1”: Peptone (0.4% w/v); Yeast extract (0.4% w/v); NaHCO₃ (0.4%w/v); Pectin (from citrus, 0.4% w/v); Xylan (from beechwood, 0.4% w/v);Arabinogalactan (0.4% w/v); Casein (0.6% w/v); unmodified wheat starch(1% w/v); inulin (0.2% w/v); bile salts (0.1% w/v); L-cysteine HCl (0.1%w/v); CaCI2) (0.0002% w/v); NaCl (0.0002% w/v); K2HP04 (0.0008% w/v);KH2P04 (0.0008% w/v); MgS04 (0.0002% w/v); Hemin (0.0001% w/v);menadione (0.00002% w/v); mucin (porcine, 0.004% w/v)

In an embodiment, the culture media used in methods of the invention,e.g., standard culture media, Media 1, etc., further comprises mucin. Itwill be understood by the skilled artisan that mucin from any sourcewhich is available and affordable can be used. For example, mucin frommammalian sources, such as bovine mucin, porcine mucin, etc., may beused. In an embodiment, porcine mucin is used.

In another embodiment, there is provided herein a media supplementtermed “Liquid Gold” and its use to supplement standard laboratoryculture media to enhance growth capabilities for microbes that wereotherwise considered “unculturable,” such as, e.g., certaingastrointestinal, anaerobic microbes. “Liquid Gold” refers to theeffluent from a chemostat in which a bacterial community is grown, i.e.,the effluent forced out of the chemostat through pressure differentials.Effluent drips into sterile bottles, housed behind the chemostat, viatubing. When the bottle is full, it can be sealed and stored at +4° C.until needed. The effluent is passed through a filter, e.g., a 0.22 μmfilter, to remove bacterial cells to produce cell-free Liquid Gold,which is used to supplement culture media.

The optimal amount of Liquid Gold to be added to a culture will varydepending on experimental conditions and the microbes to be grown, andwill be determined by the skilled artisan using standard techniques. Inan embodiment, Liquid Gold is added to 1% v/v, 3% v/v, 5% v/v, 7% v/v or10% v/v. In a particular embodiment, Liquid Gold is used to supplementgrowth media at 3% v/v.

Liquid Gold is essentially a supernatant from a microbial culture andincludes a plethora of signaling molecules, growth factors, and so on.It should be understood that the composition of a Liquid Goldpreparation will depend on the microbial community from which it isproduced. Different types of Liquid Gold can thus be made by growingdifferent bacterial communities in a chemostat. For example, “NativeLiquid Gold” is produced from chemostat effluent from culturing completenative feces in a chemostat, e.g., a single-stage chemostat system, asdescribed below. “DEC Liquid Gold” is produced from chemostat effluentfrom culturing a Defined Experimental Community (such as, e.g., DEC-1)in a chemostat, e.g., a single-stage chemostat system, as describedbelow. As used herein, “DEC-1 Liquid Gold” refers to Liquid Goldproduced from chemostat effluent from culturing the DEC-1 community;“DEC-2 Liquid Gold” refers to Liquid Gold produced from chemostateffluent from culturing the DEC-2 community; and “DEC-3 Liquid Gold”refers to Liquid Gold produced from chemostat effluent from culturingthe DEC-3 community. As reported herein, fecal samples were harvestedfrom donors of different enterotypes and cultured, and we were thereforeable to produce several different types of Liquid Gold media supplement,including, e.g., DEC-1 Liquid Gold, DEC-2 Liquid Gold, DEC-3 LiquidGold, and Native Liquid Gold.

TABLE 1 Intestinal bacterial strains isolated and purified from donorstool in DEC-1. Closest species match, inferred by alignment of 16SrRNAsequence to % identity to Relative abundance GreenGenes database*closest match (by biomass) in DEC-1 Acidaminococcus intestinalis 100 +++Bacteroides ovatus 99.52 + Bifidobacterium adolescentis 99.79 ++ (2different strains) 99.79 ++ Bifidobacterium longum 99.86 +++ (2different strains) 99.16 +++ Blautia producta** 96.43 + Clostridiumcocleatum 91.92 + Collinsella aerofaciens 98.73 + Dorea longicatena99.62 + (2 different strains) 99.60 + Escherichia coli 99.80 +Eubacterium desmolans 94.90 + Eubacterium eligens 98.15 +++++Eubacterium limosum 97.05 + Eubacterium rectale 99.59 +++++ (4 differentstrains) 99.60 +++++ 99.19 +++++ 99.53 +++++ Eubacterium ventriosum 100++ Faecalibacterium prausnitzii 99.17 +++++ Lachnospira pectinoshiza95.22 + Lactobacillus casei/paracasei 99.47 + Lactobacillus casei99.74 + Parabacteroides distasonis 99.45 ++ Raoultella sp. 99.40 +Roseburia faecalis 99.65 ++ Roseburia intestinalis 100 ++ Ruminococcustorques 99.15 +++ (2 different strains) 99.29 +++ Ruminococcus obeum94.89 + (2 different strains) 94.69 + Streptococcus mitis ^(Ψ) 99.79 +*Closest species match was inferred by alignment of 16SrRNA sequence toGreenGenes database; note that in some cases 16S rRNA gene sequencescould not resolve identity beyond genus, and that closest match does notinfer definitive speciation. Shaded boxes indicate strains that arelikely novel species (and in some cases, genera). Note that somerepresentative strains identify with the same species by 16S rRNA genesequence alignment, but we believe them to be different strains based ondifferences in colony morphology, antibiotic resistance patterns andgrowth rates. **Also referred to as Ruminococcus productus.^(Ψ)Identifies with Strep, mitis but is not α-hemolytic.

TABLE 2 Intestinal bacterial strains isolated and purified from donorstool in DEC-3. No. Strain Closest species^(c) % ID^(a) 1 11 TSABAdlercreutzia equolifaciens 99.76% 2 18 FAA SS Akkermansia muciniphila 100% 3 9 FAA NB Alistipes finegoldii 99.27% 4 19 D5 FAA Alistipesputredinis 97.15% 5 15 D5 FAA Alistipes shahii 99.85% 6 5 D5 FAA SSAlistipes sp.  100% 7 5 D5 FAA Bacteroides capillosus 96.98% 8 12 FAABacteroides cellulosilyticus 99.46% 9 9 D5 FAA Bacteroides eggerthii 100% 10 1 D6 FAA SS Bacteroides ovatus  100% 11 23 FAA Bacteroidesthetaiotaomicron  100% 12 1 TSAB Bacteroides uniformis  100% 13 17 BHIBacteroides vulgatus 99.85% 14 3 FAA SS AER. Bacillus circulans  100% 151 D5 FAA SS AER. Bacillus simplex 98.70% 16 1 D6 FAA Bifidobacteriumlongum  100% 17 18 D6 FAA SS Blautia hydrogenotrophica  100% 18 8 FAABlautia sp. 99.15% 19 4 TSA SS Blautia/Clostridium coccoides 99.85% 20 1D6 FAA SS AER. Brevibacillus parabrevis 97.60% 21 3 MRS SS Catabacterhongkongensis 98.65% 22 16 TSA SS Catabacter sp. 99.05% 23 10 TSABCatenibacterium mitsuokai 99.40% 24 13 D6 FAA SS Clostridium aldenense 192.04% 25 21 D6 FAA SS Clostridium aldenense 2 92.24% 26 13 D5 FAA SSClostridium asparagiforme 94.37% 27 3 D6 FAA SS Clostridium bolteae99.84% 28 6 D5 FAA Clostridium celerecrescens 94.48% 29 13 D6 FAAClostridium hathewayi 1 92.19% 30 21 FAA NB SS Clostridium hathewayi 291.28% 31 10 FAA Clostridium hathewayi 3 92.99% 32 11 FAA Clostridiumhathewayi 4 98.64% 33 6 D6 FAA SS Clostridium hylemonae 1 99.85% 34 8 D5FAA SS Clostridium hylemonae 2 97.85% 35 5 FAA SS Clostridium inocuum99.12% 36 11B D5 FAA SS Clostridium lavalense 99.08% 37 16 D5 FAA SSClostridium leptum 93.92% 38 4 TSA Clostridium orbiscindens 96.21% 39 14TSA Clostridium ramosum 96.14% 40 5 D6 FAA SS Clostridium scindens99.82% 41 16 BHI SS Clostridium staminisolvens 95.40% 42 17 D5 FAA SSClostridium sulfatireducens 96.63% 43 2 FAA SS Clostridium symbiosum99.83% 44 16 BHI Clostridium thermocellum 90.83% 45 18 D5 FAAClostridium sp. 1 99.16% 46 2 BHI SS Clostridium sp. 2 97.16% 47 20 D5FAA Clostridium sp. 3 95.51% 48 16 D6 FAA SS Clostridium sp. 4   98% 499 D5 FAA SS Clostridium sp. 5 97.88% 50 5 TSA Clostridium sp. 6 96.95%51 6 FAA Collinsella aerofaciens  100% 52 17 D5 FAA Coprococcus catus99.19% 53 1 BHI Coprococcus comes 99.70% 54 13 FAA Coprococcus eutactus96.49% 55 5 NA Dorea formicigenerans 99.49% 56 1 D5 FAA Dorealongicatena  100% 57 1 FAA SS AER. Escherichia coli  100% 58 5 TSABEubacterium biforme 98.76% 59 11 NA SS Eubacterium callanderi 98.08% 6019 FAA NB SS Eubacterium dolichum 93.23% 61 20 FAA Eubacterium eligens96.78% 62 9 TSAB SS Eubacterium fissicatena 97.67% 63 1 BHI SSEubacterium limosum 99.25% 64 5 D6 FAA Eubacterium rectale  100% 65 13BHI Eubacterium siraeum 93.57% 66 8 MRS Eubacterium ventriosum 97.37% 6722 D6 FAA Eubacterium xylanophilum 1 97.39% 68 15 FAA SS Eubacteriumxylanophilum 2 96.53% 69 23 D6 FAA SS Eubacterium sp. 94.31% 70 5 FAA NBFaecalibacterium prausnitzii ^(b)  100% 71 24 FAAGemmiger/Subdoligranulum 98.79% formicilis/variabile 1 72 19 D5 FAA SSGemmiger/Subdoligranulum 95.18% formicilis/variabile 2 73 17 D6 FAA SSHoldemania filiformis 97.51% 74 1 FAA NB SS AER. Microbacteriumschleiferi 99.34% 75 7 FAA NB SS AER. Micrococcus luteus 97.04% 76 21 D6FAA Odoribacter splanchnicus  100% 77 24 D6 FAA SS Oscillibactervalericigenes 95.16% 78 6 FAA NB Oscillibacter sp. 98.74% 79 16 FAAParabacteroides gordonii 99.81% 80 6 D6 FAA Parabacteroides merdae  100%81 10 D5 FAA SS Parasutterella excrementihominis  100% 82 22 FAAPhascolarctobacterium sp. 99.85% 83 10 D5 FAA Roseburia faecalis 199.84% 84 9 D6 FAA Roseburia faecalis 2 96.76% 85 9A BHI Roseburiahominis 99.04% 86 17 TSA Roseburia intestinalis  100% 87 11 TSARoseburia sp. 95.07% 88 23 D5 FAA Ruminococcus albus 96.96% 89 6 FAA NBSS Ruminococcus bromii 1  100% 90 17 FAA SS Ruminococcus bromii 2 92.83%91 17 TSAB Ruminococcus lactaris 94.46% 92 2 FAA NB Ruminococcus luti98.91% 93 15 TSA Ruminococcus obeum 99.06% 94 4 FAA Ruminococcus torques1 99.27% 95 11 FAA Ruminococcus torques 2  100% 96 8 D6 FAA SSRuminococcus torques 3 96.47% 97 9B D6 FAA SS Ruminococcus torques 491.94% 98 13 FAA NB Ruminococcus torques 5 91.47% 99 5 BHI Ruminococcussp. 1 94.32% 100 11 FAA NB Ruminococcus sp. 2 98.04% 101 4 D6 FAA SSRuminococcus sp. 3 97.05% 102 4 FAA SS AER. Staphylococcus epidermidis99.82% 103 1 FAA NB SS Streptococcus mitis  100% 104 11 FAA NB SSStreptococcus thermophilus  100% 105 12 D6 FAA SS Synergistes sp. 95.83%106 16 D5 FAA Turicibacter sanguinis  100% ^(a)% ID for each species wasdetermined using the 16S rRNA gene database, Green Genes. Average lengthof sequences used to obtain identification was 550 nucleotides. (GreenGenes BLAST interface to 16S data; accessible via lbl website) ^(b) Thestrain Faecalibacterium prausnitzii 5 FAA NB requires Liquid Gold forgrowth. A 3% final volume of Liquid Gold produced from the chemostatwhere the donor fecal sample was cultured was used to supplement FAAplates. Growth was observed after 48 hours. ^(c)Multiple strains of thesame species are denoted by a number following the species name. Forexample, Clostridium aldenense 1 and 2 are two different strains of thesame species. Shaded boxes indicate strains that are likely novelspecies (and in some cases, genera)

TABLE 3 Intestinal bacterial strains isolated and purified from donorstool in DEC-2. Closest species match, inferred by alignment of 16SrRNAsequence to GreenGenes database* Acetobacterium sp. Acidaminococcusintestinalis Anaerostipes hadrus Atopobium minutum Bacteroides fragilisBacteroides ovatus Bacteroides vulgatus Bifidobacterium adolescentis (2different strains) Bifidobacterium longum (2 different strains) Blautiacoccoides Blautia producta Clostridium aldenense Clostridium citroniaeClostridium cocleatum Clostridium hathewayi Clostridiumlactatifermentans Clostridium orbiscindens Collinsella aerofaciens Dorealongicatena (2 different strains) Escherichia coli Eubacterium desmolansEubacterium eligens Eubacterium fissicatena Eubacterium limosumEubacterium rectale (4 different strains) Eubacterium sp. (unclassified)(3 different strains) Eubacterium ventriosum Faecalibacteriumprausnitzii Lachnospira pectinoshiza Lactobacillus casei Lactobacillusparacasei Parabacteroides distasonis Raoultella sp. Roseburia faecalisRoseburia hominis Roseburia intestinalis Roseburia inulinivoransRuminococcus torques (2 different strains) Ruminococcus obeum (2different strains) Streptococcus mitis *Closest species match wasinferred by alignment of 16S rRNA sequence to GreenGenes database; notethat in some cases 16S rRNA gene sequences could not resolve identitybeyond genus, and that closest match does not infer definitivespeciation. Shaded boxes indicate strains in DEC-2 that are NOT inDEC-1.

Liquid Gold can be stored at 4° C. for weeks at a time without losingits capacity to support microbial growth, indicating that Liquid Gold'sability to support or promote growth is stable and can be preserved fora prolonged period of time. Liquid Gold can also be frozen and preservedfor future use.

In an embodiment, DEC-1 Liquid Gold is provided herein. In anotherembodiment, DEC-2 Liquid Gold is provided herein. In yet anotherembodiment, DEC-3 Liquid Gold is provided herein. In another embodiment,Native Liquid Gold is provided herein. It should be understood thatLiquid Gold can be produced from culturing many different fecal samplesand/or combinations of the human intestinal strains provided herein, andthat such types of Liquid Gold are encompassed by the present invention.For example, Liquid Gold may be produced by culturing at least one, atleast three, at least five, at least 8, at least 10, at least 15, or atleast 25 of the bacterial strains listed in Table 1, Table 2 or Table 3.In an embodiment, Liquid Gold is produced by culturing all of thestrains listed in Table 1, Table 2 or Table 3. In another embodiment,Liquid Gold is produced by culturing some of the strains listed in Table1, Table 2 or Table 3.

In a further embodiment, methods of using Liquid Gold to support and/orenhance growth of microbes, e.g., human intestinal anaerobic microbes,are provided herein. In an embodiment, Liquid Gold is used to supplementstandard laboratory culture media in a liquid culture, e.g., in achemostat. For example, Liquid Gold may be added to culture media beforeculturing begins, or during culturing of microbes. Alternatively, LiquidGold may be added to plates, e.g., solid media in a dish such as a petridish, to support or enhance growth of microbes on the solid media. Itwill be understood that many variations are possible and are encompassedby the present invention.

In an embodiment, Liquid Gold is made by growing bacterial communitiesusing the culture media referred to herein as “Media 1”, prepared asfollows:

Media 1 is prepared in the following steps (for 2 L):

Mixture 1: The following reagents are dissolved in 1800 mL of distilledwater (ddH₂O): peptone water, 4 g (Oxoid Limited); Yeast extract, 4 g(Oxoid Limited); NaHCO₃, 4 g (Sigma); CaCI₂), 0.02 g (Sigma); Pectin(from citrus), 4 g (Sigma); Xylan (from beechwood), 4 g (Sigma);Arabinogalactan, 4 g (Sigma); Starch (from wheat, unmodified), 10 g(Sigma); Casein, 6 g (Sigma); inulin (from Dahlia tubers), 2 g (Sigma);NaCl, 0.2 g (Sigma). Water (ddH₂O) is added to 1900 mL, as the volume isreduced to 1800 mL after autoclaving. The mixture is sterilized byautoclaving at 121° C. for 60 min and allowed to cool overnight.

Mixture 2: The following reagents are dissolved in 100 mL of distilledwater (Mixture 2A): K₂HP0₄, 0.08 g; KH₂P0₄, 0.08 g; MgS0₄, 0.02 g;Hemin, 0.01 g; Menadione, 0.002 g. Bile salts (1 g) is dissolved in 50mL of distilled water (Mixture 2B). L-cysteine HCl (1 g) is alsodissolved in 50 mL of distilled water (Mixture 2C). After Mixtures 2Band 2C dissolve they are added to Mixture 2A resulting in the formationof a fine white precipitate. This precipitate is then dissolved by thedrop-wise addition of 6M KOH until a clear, brown solution is formed(Mixture 2). This mixture (200 mL total volume) is sterilized byfiltering through a 0.22 μm filter.

Culture media (“Media 1”): Mixture 2 (0.2 L) is aseptically added tomixture 1 (1.8 L), in order to reach the final volume of 2 L. To preventfuture foaming, 5 mL of antifoam B silicone emulsion (J. T. Baker) isaseptically added to each 2 L bottle of media.

In an embodiment, mucin is added to Media 1 (to make “Media 1+mucin”).In this embodiment, mixture 1 is prepared by adding 1600 mL of ddH₂Obefore autoclaving. The mucin addition is prepared by dissolving 8 gmucin (e.g., from porcine stomach, type II) in 200 mL of ddH₂O, andautoclaved for 20 minutes. Mixture 2 is prepared as described above.After autoclaving, mixture 2 (0.2 L) and the mucin solution (0.2 L) areaseptically added to mixture 1 (1.6 L), in order to reach the finalvolume of 2 L. Again, 5 mL of antifoam B silicone emulsion isaseptically added to each 2 L bottle of media.

In another embodiment, Liquid Gold is made by growing bacterialcommunities using standard culture media, many of which are known in theart. In an embodiment, mucin is added to the culture media. For example,mucin may be added at a concentration of 1-10%, e.g., 4 g/L. The amountof mucin to be used will vary depending on culture conditions and isdetermined by the skilled artisan based on common general knowledge androutine methods.

In an embodiment, in order to produce Liquid Gold, a 10% w/v fecalslurry supernatant (referred to herein as a “10% inoculum) is culturedin a chemostat. In another embodiment, in order to produce Liquid Gold,a 20% w/v fecal slurry supernatant (referred to herein as a “20%inoculum) is cultured in a chemostat. In other embodiments, 5-25%, 5%,10%, 15%, 20% or 25% inoculums are cultured in a chemostat, e.g., asingle-stage chemostat system, to produce Liquid Gold. It should beunderstood that any % inoculum can be used to inoculate a chemostatvessel to produce Liquid Gold, as long as a sufficient amount of thefecal sample is present to seed the vessel, and the resulting fecalslurry is not too thick or viscous to work with.

In an embodiment, in order to produce Liquid Gold, a chemostat system isused where the growth medium is continuously fed into the chemostatvessel at a rate of 400 mL/day (16.7 mL/hour) to give a retention timeof 24 hours, a value set to mimic the retention time of the distal gut(Cummings, J. H. et al., Gut (1976), 17:210-18). In another embodiment,the growth medium is continuously fed into the chemostat at a rate ofabout 148 mL/day (6.2 mL/hour) to give a retention time of 65 hours. Inan embodiment, Liquid Gold is produced from a chemostat having a systemretention time of about 20 to about 70 hours, about 20 hours, about 22hours, about 24 hours, about 26 hours, about 28 hours, or about 30hours, about 40 hours, about 50 hours, about 60 hours, about 65 hours,or about 70 hours.

We report herein that several novel bacterial species from the human guthave been cultured by supplementation of culture media with Liquid Gold(e.g., 3% v/v Liquid Gold). Several bacterial species have been isolatedfor the first time using the methods provided herein, including usingLiquid Gold. In an aspect, there is provided herein a method ofculturing Faecalibacterium prausnitzii, wherein the growth media issupplemented with Liquid Gold. In some embodiments, there is providedherein a method of culturing strains which have not been previouslyisolated, e.g., Clostridium aldenense 1, Clostridium aldenense 2,Clostridium hathewayi 1, Clostridium hathewayi 2, Clostridium hathewayi3, Clostridium thermocellum, Ruminococcus bromii 2, Ruminococcus torques4, Ruminococcus torques 5, Clostridium cocleatum (e.g., Clostridiumcocleatum 21 FAA1), Eubacterium desmolans (e.g., Eubacterium desmolans48FAA1), Eubacterium limosum 13LG, Lachnospira pectinoshiza,Ruminococcus productus (e.g., Ruminococcus productus 27FM), Ruminococcusobeum (e.g., Ruminococcus obeum 11FM1), Blautia producta, and/orClostridium thermocellum, wherein the growth media is supplemented withLiquid Gold.

In an aspect, there are provided herein certain isolated bacterialstrains, which have not been previously isolated. In an embodiment,there is provided herein an isolate of Faecalibacterium prausnitzii. Inanother embodiment, there is provided herein an isolate of Clostridiumaldenense 1, Clostridium aldenense 2, Clostridium hathewayi 1,Clostridium hathewayi 2, Clostridium hathewayi 3, Clostridiumthermocellum, Ruminococcus bromii 2, Ruminococcus torques 4,Ruminococcus torques 5, Clostridium cocleatum (e.g., Clostridiumcocleatum 21 FAA1), Eubacterium desmolans (e.g., Eubacterium desmolans48FAA1), Eubacterium limosum 13LG, Lachnospira pectinoshiza,Ruminococcus productus (e.g., Ruminococcus productus 27FM), Ruminococcusobeum (e.g., Ruminococcus obeum 11FM1), Blautia producta, or Clostridiumthermocellum.

Culture conditions for exemplary bacterial strains isolated usingmethods provided herein are given in Table 4.

TABLE 4 Culture conditions for anaerobic strains isolated from a humanfecal sample. Growth media used Relative for synthetic stool growthStrain Closest species Colony morphology preparations' rate2 18 FAAEubacterium rectale Small, white/ FAA + 5% DSB +++ translucent 10 FAADorea longicatena Small/medium, FAA + 5% DSB +++ opaque, somewhat mucoid42 FAA 1 Dorea longicatena Medium, opaque, FAA + 5% DSB +++ pitting 31FAA 1 Roseburia intestinalis Medium, opaque FAA + 5% DSB + +++ 3% LG 6MRS Lactobacillus Medium, white, FAA + 5% DSB +++ casei/paracasei sticky1 FAA Eubacterium rectale Pinpoint, FAA + 5% DSB +++ opaque/white 27 FMRuminococcus Small, white, dry FAA + 5% DSB + productus 30 FAARuminococcus Small, white, dry FAA+5%DSB +++ torques 2 MRS Ruminococcusobeum Medium, white/ FAA + 5% DSB + opaque, sticky 6 FM 1 Eubacteriumrectale Medium, white/ FAA + 5% DSB + +++ opaque, sticky 3% LG 2 FAABifidobacterium Small, brown, FAA + 5% DSB +++ longum translucent,metallic sheen, sticky 39 FAA 1 Roseburia faecalis Medium, white/ FAA +5% DSB + +++ opaque, pitting 3% LG 14 LG 2 Acidaminococcus Large, whiteFAA + 5% DSB +++ intestinalis 5 FM Parabacteroides Small, white, FAA +5% DSB +++ distasonis translucent 21 FAA 1 Clostridium Medium, white/FAA + 5% DSB +++ cocleatum opaque, very pitting/difficult to scrape,sticky 20 MRS 1 Bifidobacterium Pin, brown/opaque, FAA + 5% DSB +++adolescentis slight metallic sheen, sticky 48 FAA 1 EubacteriumPinpoint, white/ FAA + 5% DSB + desmolans opaque, sticky 5 MM 1Bacteroides ovatus Small, white/ FAA + 5% DSB +++ translucent 4 FM 1Bifidobacterium Pinpoint, translucent, FAA + 5% DSB +++ longum yellow,dry, pitting, metallic sheen, sticky 11 FM 1 Ruminococcus obeum Small,white/ FAA + 5% DSB ++ opaque, translucent F1 FAA 1 Eubacterium eligensPinpoint, FAA + 5% DSB + + pink/purple/opaque 3% LG 25 MRS 1Lactobacillus casei Small, white/ FAA + 5% DSB +++ opaque, sticky 13 LGEubacterium limosum Small, off- FAA + 5% DSB + +++ white/opaque 3% LG 9FAA Ruminococcus Small, white/ FAA + 5% DSB +++ torques opaque,translucent 47 FAA Eubacterium Sticky, small FAA + 5% DSB +++ ventriosum3 FM 2 Collinsella Pinpoint, FAA + 5% DSB +++ aerofaciens white/opaque,translucent, dry 11 FAA 1 Bifidobacterium Small, yellow/ FAA + 5% DSB+++ adolescentis opaque, mucoid 34 FAA 1 Lachnospira Pinpoint, yellowFAA + 5% DSB + ++ pectinoshiza 3% LG 40 FAA Faecalibacterium Pinpoint,transparent FAA + 3% LG +++ prausnitzii 29 FAA 1 Eubacterium rectalesmall, white/ FAA + 5% DSB +++ opaque, translucent 1 FAA: Fastidiousanaerobe agar, commercially available as Lab90; DSB: Defibrinated sheepblood, commercially available; LG: Liquid Gold, a clarified, filteredeffluent supernatant from chemostat communities seeded from healthyfecal communities, required by a number of synthetic stool strains foroptimal growth. 2Relative growth rate; on average plates were incubatedfor 3 days at 37° C. under anaerobic conditions.

As used herein, “anaerobic” bacteria refers to bacteria which arefacultatively anaerobic as well as bacteria which are strictlyanaerobic.

As used herein, “standard culture media” refers to common and/orcommercially available growth media for microorganisms, such as nutrientbroths and agar plates, of which many variations are known in the art.Standard culture media generally contains at least a carbon source forbacterial growth, e.g., a sugar such as glucose; various salts which arerequired for bacterial growth, e.g., magnesium, nitrogen, phosphorus,and/or sulfur; and water. Non-limiting examples of standard culturemedia include Lysogeny broth (LB), A1 broth, and culture media describedherein. Standard culture media for use in methods provided herein willbe selected by a skilled artisan based on common general knowledge. Theterms “standard culture media” and “standard laboratory culture media”are used interchangeably herein.

As used herein, the terms “pure isolate,” “single isolate” and “isolate”are used interchangeably to refer to a culture comprising a singlebacterial species or strain, e.g., grown axenically, in isolation fromother bacterial species or strains.

For strains listed in the tables herein, the closest bacterial specieswas determined using the 16S rRNA full-length sequences, which werealigned with the NAST server (DeSantis, T. Z. Jr. et al., Nucleic AcidsRes., 34:W394-W399 (2006)) and were then classified using the GreenGenesclassification server (DeSantis, T. Z. Jr. et al., Appl. Environ.Microbiol., 72:5069-5072 (2006)), as described below. The most specificname in the GreenGenes classification was used and we report the DNAmaximum likelihood score for each classification.

EXAMPLES

The present invention will be more readily understood by referring tothe following examples, which are provided to illustrate the inventionand are not to be construed as limiting the scope thereof in any manner.

Unless defined otherwise or the context clearly dictates otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. It should be understood that any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention.

Materials and Methods Single-Stage Chemostats and Inoculation

We developed a single-stage chemostat vessel to model the human distalgut microbiota by modifying a Multifors fermentation system (Infors,Switzerland; shown in FIG. 1). Conversion from a fermentation systeminto a chemostat was accomplished by blocking off the condenser andbubbling nitrogen gas through the culture. The pressure build up forcedthe waste out of a metal tube (formerly a sampling tube) at a set heightand allowed for the maintenance of a 400 mL working volume.

Throughout the duration of the experiment, the vessels were keptanaerobic by bubbling filtered nitrogen gas (Praxair) through theculture. Temperature (37° C.) and pH (set to 7.0; usually fluctuatedaround 6.9 to 7 in the culture) were automatically controlled andmaintained by the computer-operated system. The system maintained theculture pH using 5% (v/v) HCl (Sigma) and 5% (w/v) NaOH (Sigma). Thegrowth medium was continuously fed into the vessel at a rate of 400mL/day (16.7 mL/hour) to give a retention time of 24 hours, a value setto mimic the retention time of the distal gut (Cummings, J. H. et al.,Gut (1976), 17:210-18). Another retention time of 65 hours (−148 mL/day,6.2 mL/hour) was also tested to determine the effect of retention timeon the composition of the chemostat community.

Since the growth medium contained components which cannot survivesterilization by autoclaving (see below), the vessels were autoclavedwith 400 mL of ddH₂O. During autoclaving, the waste pipes were adjustedso the metal tube reached the bottom of the vessel. Once the vesselcooled it was fitted to the rest of the computer operated unit, filterednitrogen gas was bubbled through the water to pressurize and drain thevessel. The waste pipe was then raised to the working volume (400 mL)and 300 mL of sterile media was pumped into the vessel. The vessel wasthen left stirring, heating, and degassing overnight. To check forcontamination within the vessel, each vessel was aseptically sampled andplated out (both aerobically and anaerobically) on fastidious anaerobeagar (FAA) supplemented with 5% defibrinated sheep blood. This procedurewas repeated one day before inoculation and immediately prior toinoculation to ensure contamination was avoided.

Collection and Preparation of Fecal Inocula

Fresh fecal samples were provided by a healthy female donor (42 yearsold, with no history of antibiotic use in the 10 years prior to stooldonation; “Donor 6”) or by a healthy male donor (43 years old, with nohistory of antibiotic use in the 6 years prior to stool donation; “Donor5”). Other donors also provided fecal samples (e.g., Donor 1, Donor 2,etc.). All donors were healthy subjects from 38 to 43 years of age withno recent history of antibiotic treatment. Research Ethics Board (REB)approval was obtained for fecal collection and use in these experiments.

To prepare the inoculum, freshly voided stool samples were collected andimmediately placed in an anaerobic chamber (in an atmosphere of 90% N₂,5% CO₂ and 5% H₂). A 10% (w/v) fecal slurry was immediately prepared bymacerating 5 g of fresh feces in 50 mL of anaerobic phosphate bufferedsaline (PBS) for 1 minute using a stomacher (Tekmar Stomacher LabBlender, made by Seward). The resulting fecal slurry was centrifuged for10 minutes at 1500 rpm to remove large food residues. The resultingsupernatant was used as the inoculum for this study. The 10% originalw/v fecal slurry supernatant is referred to herein as the “10%inoculum”. We also compared a 10% inoculum to a 20% (w/v) inoculum todetermine whether any differences existed between these twoconcentrations. The 20% (w/v) inoculum was prepared in the same manneras the 10% inoculum except that 10 g of feces was added to 50 mL ofanaerobic PBS prior to homogenization. Again, the inoculum derived fromthe 20% original w/v fecal slurry supernatant is referred to herein asthe “20% inoculum”.

Inoculation Process

To give a final working volume of 400 mL, 100 mL of 10% inocula wasadded to the 300 mL of sterile media in each vessel. Since the thicknessof the fecal inoculum made it difficult to seed the vessel through theseptum using a needle, the inoculum was added to the vessel through thewaste pipe using a syringe. Immediately following inoculation the pHcontrols were turned on so the vessel pH was adjusted to and maintainedat a pH of about 6.9 to 7.0. During the first 24 hours post-inoculationthe communities were grown in batch culture to allow the community toadjust from in vivo to in vitro conditions and avoid culture washout.During this period the vessels were heated, degassed and stirred withcontinuous pH adjustment. After this 24 hour period the feed pumps wereturned on and the vessels were run as chemostats. Fresh culture mediumwas added continuously and waste was continuously removed.

In the chemostat, culture conditions and media supply were maintainedconstant. The chemostat system was generally set with a retention timeof 24 hours to mimic distal gut transit time.

Preparation of the Growth Medium

A culture growth medium for the chemostat was developed based on mediaused in previous studies attempting to mimic the human gut (Gibson, G.R. et al., Appl. Environ. Microbiol., 54(11):2750-5, 1988; Lesmes, U. etal., J. Agric. Food Chem., 56: 5415-5421, 2008). Due to the large amountof medium used by each vessel, medium was prepared in 2 L volumes. Theculture medium was prepared in the following steps (for 2 L):

Mixture 1: The following reagents were dissolved in 1800 mL of distilledwater (ddH₂O): peptone water, 4 g (Oxoid Limited); Yeast extract, 4 g(Oxoid Limited); NaHCO₃, 4 g (Sigma); CaCI₂), 0.02 g (Sigma); Pectin(from citrus), 4 g (Sigma); Xylan (from beechwood), 4 g (Sigma);Arabinogalactan, 4 g (Sigma); Starch (from wheat, unmodified), 10 g(Sigma); Casein, 6 g (Sigma); inulin (from Dahlia tubers), 2 g (Sigma);NaCl, 0.2 g (Sigma). Water (ddH₂O) was added to 1900 mL, as the volumeis reduced to 1800 mL after autoclaving. The mixture was sterilized byautoclaving at 121° C. for 60 min and allowed to cool overnight.

Mixture 2: The following reagents (all purchased from Sigma) weredissolved in 100 mL of distilled water (Mixture 2A): K₂HP0₄, 0.08 g;KH₂P0₄, 0.08 g; MgS0₄, 0.02 g; Hemin, 0.01 g; Menadione, 0.002 g. Bilesalts (1 g) was dissolved in 50 mL of distilled water (Mixture 2B).L-cysteine HCl (1 g) was also dissolved in 50 mL of distilled water(Mixture 2C). After Mixtures 2B and 2C dissolved they were added toMixture 2A resulting in the formation of a fine white precipitate. Thisprecipitate was then dissolved by the drop-wise addition of 6M KOH untila clear, brown solution was formed (Mixture 2). This mixture (200 mLtotal volume) was sterilized by filtering through a 0.22 μm filter.

Culture media (“Media 1”): Mixture 2 (0.2 L) was aseptically added tomixture 1 (1.8 L), in order to reach the final volume of 2 L. To preventfuture foaming, 5 mL of antifoam B silicone emulsion (J. T. Baker) wasaseptically added to each 2 L bottle of media. The media was stored at4° C. until use for a maximum of two weeks. A bit of media was platedout on FAA (aerobically and anaerobically) the day before adding tochemostat and immediately after taking off the chemostat, to check forcontamination.

The media was pumped into each vessel using a peristaltic pump whosespeed is controlled by the computer-operated system. To pump media fromthe bottles into the vessel, standard GL-45 glass bottle lids (VWR) hadholes drilled into them to fit two stainless steel metal tubes. WhenMixture 1 was prepared, the media bottle had all the required siliconetubing and 0.22 μm filters attached (see FIG. 1).

Each vessel was fed from one media bottle with a 2 L volume of media.Since the tubing which supplied the media to the vessel was also changedas each media bottle was changed, this helped to prevent back-growth ofbacteria from the vessel into the sterile media reservoir. Each mediabottle was plated out on supplemented FAA and grown both aerobically andanaerobically before each bottle was added to the chemostat and aftereach bottle was removed from the chemostat.

We compared our culture media (Media 1) to a media previously describedin the literature (Walker, A. W. et al., Appl. Environ. Microbiol., 71(7): 3692-700, 2005), since pH and peptide supply can alter bacterialpopulations and short-chain fatty acid ratios within microbialcommunities from human colon. This media was prepared using a similarmethod as was used to prepare our media, only the composition of the twomixtures changed. The chemostat media described in Walker et al. wasprepared in the following steps (for 2 L):

Mixture 1: The following reagents were dissolved in 1800 mL of distilledwater: peptone water, 12 g; NaHCO₃, 6.4 g; pectin (from citrus), 1.2 g;xylan (from beechwood), 1.2 g; arabinogalactan, 1.2 g; starch (wheat,unmodified), 10 g; casein hydrolysate, 12 g; amylopectin, 1.2 g. Thismixture was sterilized in an autoclave at 121° C. for 60 min.

Mixture 2: L-cysteine HCl (1 g) was dissolved in 100 mL of distilledwater (Mixture 2A). Bile salts (1 g) were dissolved in 100 ml ofdistilled water (Mixture 2B). Mixture 2B was added to Mixture 2Aresulting in the formation of a fine white precipitate. The pH of thesolution was then adjusted by the drop-wise addition of 6M KOH until aclear, colourless solution was formed. This mixture (200 mL totalvolume) was sterilized by filtering through a 0.22 μm filter.

Chemostat media: Mixture 2 (0.2 L) was aseptically added to mixture 1(1.8 L), in order to reach the final volume of 2 L. To prevent futurefoaming, 5 mL of antifoam B silicone emulsion was aseptically added toeach 2 L bottle of media. The media was stored at 4° C. until use for amaximum of two weeks.

To determine whether the addition of mucin to our culture media(Media 1) had an effect on the composition and structure of healthydistal gut communities, we compared one vessel fed with our culturemedia (without mucin) to two vessels fed with our culture media(containing mucin). The chemostat media with mucin was prepared in asimilar manner as our culture media without mucin, with a couple ofchanges. Firstly, mixture 1 was prepared by adding 1600 mL of ddH₂Obefore autoclaving. The mucin addition was prepared by dissolving 8 gmucin (from porcine stomach, type II) in 200 mL of ddH₂O, and autoclavedfor 20 minutes. Mixture 2 was prepared as described above. Afterautoclaving, mixture 2 (0.2 L) and the mucin solution (0.2 L) wereaseptically added to mixture 1 (1.6 L), in order to reach the finalvolume of 2 L. Again, 5 mL of antifoam B silicone emulsion wasaseptically added to each 2 L bottle of media. The media was also storedat 4° C. until use for a maximum of two weeks.

Sampling

Each chemostat vessel was sampled daily by removing 4 mL of culturethrough the septum using a sterile needle and syringe. Samples weretransferred into two screw-capped tubes which were stored at −80° C. toarchive. During weekdays, 10 drops of antifoam B silicone emulsion wasadded through the septum by a syringe and needle at 9 am and 5 pm (20drops per day total). On weekends, 20 drops of antifoam was added toeach vessel around 12 pm. This amount of antifoam added to each vesseldaily (in conjunction with the amount of antifoam present in the media)was sufficient to prevent foaming in our system using a 24 hourretention time.

DNA Extraction

The DNA used for DGGE analysis was extracted using a protocol involvinga combination of bead beating, the Omega Bio-Tek E.Z.N.A.® Stool DNAKit, and the Promega Maxwell®16 DNA Purification Kit. The first half ofthe protocol follows the June 2009 revision of the E.Z.N.A.® Stool DNAKit protocol with a few alterations. Briefly, we added 200 μL of liquidchemostat or fecal sample, 300 μL of SLX buffer from the E.Z.N.A. kit,10 μL of 20 mg/mL proteinase K (in 0.1 mM CaCI₂)) and 200 mg of glassbeads to a screw-capped tube and bead beat for 4×45 seconds (3 minutestotal). The samples were then incubated at 70° C. for 10 minutes, 95° C.for 5 minutes and on ice for 2 minutes. Next we added 100 μL of BufferP2 from the E.Z.N.A. kit to each tube and vortexed them for 30 seconds.This was followed by incubation on ice for 5 minutes and centrifugationat 14500×g for 5 minutes. The supernatant was then transferred into anew 1.5 mL tube and 200 μL of HTR reagent from the E.Z.N.A. kit wasadded to each tube using wide bore tips. The samples were then vortexedfor 10 seconds and incubated at room temperature for 2 minutes. Thetubes were then centrifuged at 14500×g for 2 minutes and the supernatantwas transferred into Maxwell cartridges. The remainder of the DNAextraction protocol was carried out as described in the Maxwell®16 DNAPurification Kit protocol (Promega).

PCR and DGGE

The V3 region (339-539 bp, Escherichia coli numbering) of the 16S rRNAgene was amplified using primers HDA1 and HDA2-GC (Walter, J. et al.,Appl. Environ. Microbiol., 66(1): 297-303, 2000). The PCR master mixconsisted of Tsg DNA polymerase (Bio Basic) and 1× Thermopol buffer(with 2 mM MgS0₄, NEB), using DNA (extracted as described above) as atemplate. The cycling conditions were as follows: 92° C. for 2 min (92°C. for 1 min, 55° C. for 30 sec, 72° C. for 1 min)×35; 72° C. for 10min. Three identical 50 μL PCR reactions were set up for each sampleusing 2 μL of DNA template. Each sample was concentrated using the EZ-10Spin Column PCR Products Purification Kit (Bio Basic) by loading allthree PCR reactions from each sample onto the same column and eluting in45 μL of warm HPLC grade water. Once the PCR reactions were concentrated10 μL of DGGE loading dye (0.05 g bromophenol blue in 10 ml 1×TAE) wasadded to each sample.

A DGGE ladder created from five laboratory strains was used to normalizethe gel. This ladder consisted of V3 DGGE PCR reactions from laboratorystrains 1/2/53 (Coprobacillus), 30/1 A (Enterococcaceae), 5/2/43 FAA(Veillonella), 1/1/41 A1 FAA CT2 (Peptostreptococcaceae), and 7/6/55BFAA (Propionibacterium). DNA from these strains was extracted using themethod described by Strauss et al. (Strauss, J. et al., Anaerobe, 14(6):301-9, 2008). The PCR reactions used to generate the amplicons toconstruct the ladder were prepared as described above, except 1 PCRreaction was prepared per stain. The five different PCR reactions werepooled and 62.5 μL of DGGE loading dye was added to the mixture.

The protocol used for DGGE analysis was developed based on a protocolusing the DCode System (Bio-Rad Laboratories, Hercules, Calif., USA)described by Muyzer et al. (Muyzer, G. et al., Antonie Van Leeuwenhoek,73(1): 127-41, 1998). 40 μL of the PCR/dye mixture was loaded onto eachlane of the gel. The polyacrylamide gels consisted of 8% (v/v)polyacrylamide (37.5:1 acrylamide/bisacrylamide) in 0.5×TAE (TAE is Trisbase, acetic acid and EDTA buffer, made using the following recipe: Trisbase [tris(hydroxymethyl)aminomethane] (0.048% w/v); glacial acetic acid(17.4M) (0.011% v/v); EDTA disodium salt (0.0037% w/v)). The ampliconswere separated using a denaturating gradient of 30-55%. Electrophoresiswas performed in 0.5×TAE buffer at a constant temperature of 60° C. for5 h at 120 V. Gels were stained for 10 minutes in ethidium bromidesolution (in 1×TAE, Sigma Aldrich) and destained for 10 minutes inddH₂O. Images were captured using the SynGene G-Box gel documentationsystem and GeneSnap software (version 6.08.04). The gels were normalizedfor saturation while the images were captured.

DGGE Statistical Analyses

DGGE gel images were analyzed using the Syngene GeneTools software(version 4.01.03, Perkin Elmer). The image background was subtractedusing rolling disc subtraction with a radius of 30 pixels. Lanes weremanually detected and set on each gel image.

Analysed bands were both automatically and manually detected for eachprofile. The profiles were matched using the “profile” type under thematching parameters menu with a set tolerance of 1%. Dendrograms weredrawn using the Unweighted Pair Group Method with Arithmetic Mean(UPGMA). Profile comparison resulted in an automatically generatedsimilarity matrix and dendrogram. Similarity index values range from 0to 1, with values of 0 indicating two profiles have no bands in common,while values of 1 indicate the two profiles have identical bandingpatterns. Percent similarity values were calculated by multiplying thesimilarity index value by 100.

Comparing two vessels on day (x): The similarity of two vessels wasdetermined plotting the % similarity of V(x) vs. V(y) against the daythe sample was taken. This analysis was carried out for samples takenevery two days beginning at Day 0.

Community dynamics: Community dynamics represents the changes within acommunity over a fixed time frame. Moving window analysis was performedby plotting the % similarity between consecutive sampling points. Inthis case we chose to plot Day (x-2) vs. Day (x). We found that thistime interval was adequate and did not cause us to miss largefluctuations in the community dynamics and was in agreement withprevious literature (Possemiers, S. et al., FEMS Microbiol. Ecol.,49(3): 495-507, 2004). This analysis resulted in the generation of agraph which was used to assess the stability of the community as well asto determine how long it took the vessel to reach steady state. Anexample of a moving window correlation plot illustrating communitydynamics is shown in FIG. 13 c.

The rate of change (Δt) can then be calculated as 100—the average of therespective moving window curve data points (Marzorati, M. et al.,Environ. Microbiol., 10(6): 1571-81, 2008). The larger the changebetween the profiles of the consecutive sampling points the higher theΔt value. However, since an initial stabilization period is noted as thecommunity transitions from an in vivo to an in vitro environment, valuesmay vary depending on the period chosen (Marzorati, M. et al., Environ.Microbiol., 10(6): 1571-81, 2008). According to two papers by Wittebolleet al. (Marzorati, M. et al., Environ. Microbiol., 10(6): 1571-81, 2008;Wittebolle, L. et al., J. Appl. Microbiol., 107(2):385-94, 2009), a lowΔt value ranges from 0-5%, a medium value ranges from 5-15%, and a highvalue is 15+%. Steady state is reached once the curve of the graphremains above the set threshold. We considered our chemostat communitiesto be stable (at steady state) once a low Δt value (0-5%) was maintainedby the community.

Shannon Diversity Index

The Shannon index is a commonly used mathematical measure of communitydiversity which takes into account both species richness (number ofspecies present) and evenness (relative species abundance). The Shannondiversity index (H′) is calculated as shown below (Marzorati, M. et al.,Environ. Microbiol., 10(6): 1571-81, 2008):

$\begin{matrix}s \\{H^{\prime} = {\text{-}{\sum( {p_{i}{{In}p}_{i}} )}}} \\{i = 1}\end{matrix}$

where:

-   -   H′=the value of the Shannon diversity index    -   p_(i)=the proportion of the ith species    -   In=the natural logarithm of p_(i)    -   s=total number of species in the community (richness)    -   Σ=sum from species 1 to species s.        The minimum value of the Shannon index is zero, which is equal        to the value of H′ for a community with a single species (i.e.,        a monoculture with no diversity). The H′ value increases as        community richness and evenness increase. Because of this, an        increase in H′ may be the result of an increase in species        richness, an increase in species evenness, or an increase in        both. This is a flaw in the index and the reason that care        should be taken when using this measure of diversity. H′ values        have been found to range from 1.5 (low species richness and        evenness) to 3.5 (high species evenness and richness) in natural        systems (MacDonald, G. M., 2003, Biogeography: Space, Time and        Life, John Wiley & Sons, Inc., U.S.A., pg 409). However, we find        it more important to use the Shannon index to measure and track        changes in the diversity of samples as compared to the original        fecal inoculum (Gafan, G. P. et al., J. Clin. Microbiol., 43(8):        3971-8, 2005).

Range-Weighted Richness

Range-weighted richness (Rr) is a measure of community richness thattakes the percentage of denaturant needed to describe the diversity ofthe community into account when analyzing DGGE gels (Marzorati, M. etal., Environ. Microbiol., 10(6):1571-81, 2008). Rr is calculated asshown below:

Rr=N ² ×D _(g)

where:

-   -   Rr=Range-weighted richness    -   N=total number of bands in the pattern    -   D_(g)=denaturing gradient comprised between the first and last        band of the pattern.        Low Rr values are less than 10, medium Rr values range from 10        to 30, and high Rr values are greater than 30 (Marzorati, M. et        al., Environ. Microbiol., 10(6):1571-81, 2008).

Shannon's Equitability

Community evenness is the degree to which the numbers of individuals areevenly divided between the different species of the community. Communityevenness can be assessed by calculating Shannon's equitability (EH;Marzorati, M. et al., Environ. Microbiol., 10(6): 1571-81, 2008). E_(H)is calculated by dividing Shannon index (H′) by H_(max) (where H_(max)is InS). This is shown below as follows:

E _(H) =H′/H _(max) =H′/InS

E_(H) values range from 0-1, with a value of 0 representing completecommunity unevenness and a value of 1 representing complete communityevenness. Increases in the evenness result in an increase in communitydiversity (Pielou, E. C. 1975. Ecological diversity. Wiley, New York).

Example 1. Threshold for DGGE Analysis

A study by Possemiers et al. (Possemiers, S. et al., FEMS Microbiol.Ecol., 49(3):495-507, 2004) established a threshold stability criterionof 80% similarity (or 20% variability) for DGGE studies based onwithin-gel variability seen between identical marker lanes. According tothis study, the threshold can be used in conjunction with moving windowcorrelation analysis to monitor the dynamics of the community. Thisapproach allows us to examine the similarity of a vessel to itself overtime. The 80% similarity threshold can then be used to determine howlong it takes a vessel to rise above this cut-off and reach steadystate.

In order to apply this threshold to our studies, a similar analysis wascarried out on the marker lanes from our DGGE gels. We found an averageof 80.4±8.9% similarity, or 19.6±8.9% variability, between these markerlanes. Since these values correspond well with the values used in thePossemiers et al. study, a threshold of 80% similarity was also used forour analyses. In some cases individual within gel variation was used asa cut-off.

In the Examples below, we analyzed the colonization process in twoidentical vessels to determine whether these vessels can be run inparallel and still maintain identical communities. We also compareddifferent concentrations of fecal inocula, different compositions ofmedia, and different system retention times to optimize the operation ofour chemostat system.

Example 2. Comparison of Same Donor Over Time

The DGGE pattern of 10% inocula from Donor 2 (a 38-year old healthyfemale) over an 8 month period is shown in FIG. 2. As seen by visualinspection, the variation in the profiles seems to be due to differencesin band brightness, not the appearance or disappearance of bands. Theinocula isolated from this donor had an average correlation coefficientof 76.9±8.7%. Slight differences between profiles were shown in FIG. 2where samples showed slightly higher similarity depending on the time ofsample collection. Overall, the gut microbiota of this healthy donorremained stable over time.

The gut microbiota of this donor maintained a high diversity over time,with an average Shannon-Weaver index value of 3.39±0.08. The donor'smicrobiota also maintained a very high average range-weighted richnessat 776.5±27.7. Finally, the community evenness was stable over time,with an average Shannon equitability value of 0.82±0.02.

Example 3. Comparison of Different Donors

We used DGGE to compare fecal inocula isolated from four differenthealthy donors (from 38 to 43 years of age with no recent history ofantibiotic treatment) (FIG. 3). We found that the fecal community ofeach donor was different from the communities of other individuals (asexpected, Tannock, G. W., Eur. J. Clin. Nutr., 56 Suppl. 4:S44-9, 2002).The average correlation coefficient between the inocula from differentdonors was 74.4±8.4%.

The gut microbiota of all four donors had high diversity, with anaverage Shannon-Weaver index value of 3.42±0.04. The donor microbiotaalso maintained a very high average range-weighted richness at585.3±26.2. The community evenness between the different donors wasquite similar, with an average Shannon equitability value of 0.86±0.01.

We also used DGGE to compare two different communities, seeded by fecalsamples from two different healthy donors, each of whom donated on atleast two separate occasions. Donor 5 made two donations, about 5 monthsapart: “Run 20” was inoculated on Oct. 28, 2011, and “Run 22” wasinoculated on Mar. 23, 2012. Donor 6 made two donations about 6 monthsapart: “Run 16” was inoculated on Feb. 10, 2011, and “Run 19” wasinoculated on Aug. 3, 2011. We asked how similar are the inocula fromthe two different donors to each other; whether we would see the sameloss of diversity from each donor; and how different are the two donorsfrom each other.

Results are shown in FIGS. 10-13. FIG. 10 shows that fecal inocula usedto seed the chemostat vessels was very similar to the starting fecaldonation, and not altered significantly by the process of preparing theinoculum. FIG. 11 shows that DGGE profiles from Donors 5 and 6 wereconsistent between donations. FIG. 12 shows that, by DGGE, the fecalinocula from the same donor on two different occasions were more similarto each other than to fecal inocula from another donor. Also, the steadystate communities seeded with feces from the same donor were moresimilar to each other between chemostat runs than to communities seededwith feces from another donor. FIG. 13 shows that evenness in twovessels with the same inocula was similar to each other, stable overtime, and similar to that of the starting inocula.

Example 4. Comparison of 10% Vs. 20% Inocula

DGGE was used to assess whether a 10% or 20% inoculum was better suitedto seed a chemostat vessel by maintaining a higher diversity (FIG. 4).Within-group comparisons of the 10% inocula gave a correlationcoefficient of 98.1%, while the 20% inocula gave a correlationcoefficient of 98.4%. Between-group comparisons of the 10% and 20%inocula gave an average correlation coefficient of 97.9±0.7%. Withlittle differences between the within- and between-group values, littledifferences in the concentrations of inocula were observed.

There were no differences between the diversity of the 10% and 20%inocula. The Shannon-Weaver index values for both the 10% and 20%inocula were high, with values of 3.38 and 3.37, respectively. Also, therange-weighted richness values for both the 10% and 20% inocula werevery high, with values of 802.9 and 810.0, respectively. There were alsono differences between the evenness of the 10% and 20% inocula. The 10%and 20% inocula both had Shannon equitability values of 0.82.

Both inocula were similar to the fecal samples, with correlationcoefficients of 86.4% for the 10% inocula and 80.9% for the 20% inocula.These inocula were also similar to the respective pellets formed duringinocula preparation, with correlation coefficients of 73.7% for the 10%inoculum and 75.5% for the 20% inoculum (Table 5).

TABLE 5 Correlation coefficients for the 10% inocula and the 20%inocula. 10% inoculum 20% inoculum Feces vs. inoculum 86.4 80.9 Inoculumvs. pellet 73.7 75.5 Feces vs. pellet 86.4 85.7

Example 5. Comparison of Two Vessels Run in Parallel

DGGE was used to monitor the composition, diversity, and dynamics of twoidentical chemostat vessels (V1 and V2). Each vessel was seeded withfecal inocula from the same healthy donor to determine whether these twovessels could maintain identical communities.

The inocula used to seed each vessel were very similar to each other andimmediately after inoculation the correlation coefficients of samplestaken from each vessel was 91.3%. The composition of each vessel variedfrom each other between days 2-8, however the communities within eachvessel became more similar to each other between days 10-28, with anaverage correlation coefficient of 94.7±2.0%.

GeneTools (statistical analysis software; Syngene) only takes speciesrichness into account when calculating its similarity indices. Bothvessels were 95.6% similar to each other on day 10 based on theGeneTools analysis and therefore shared most of their bands between theprofiles. However, the vessels differed from each other in terms of thebrightness of these bands. Upon visual inspection supported by measuresof evenness, both vessels showed identical communities in terms ofbanding patterns and band brightness by day 26.

During the initial 10 days of the experiment the communities in bothvessels were unstable and had high Δt values as determined using movingwindow analysis (FIG. 5). Between days 0-10, both vessels had similarhigh Δt values, with averages of 25.1±13% for V1 and 20.8±9.0% for V2(p>0.10). Between days 24 and 28, V1 and V2 had similar low dynamics,with Δt values of 1.6±0.2% for V1 and 3.9±1.6% for V2 (p>0.10).

The community diversity was very high throughout the duration of theexperiment, however an initial drop was observed between days 0 and 10,with Shannon-Weaver index values dropping from 3.38 to 2.93 for V1 andfrom 3.38 to 3.00 for V2. This drop evened out by day 14, giving anaverage Shannon-Weaver index value of 3.03±0.06 for V1 and 2.98±0.10 forV2 between days 14 and 28 (p>0.10). The community range-weightedrichness also saw a drop between days 0 to 10, with values dropping from578.6 to 341.7 for V1 and from 582.4 to 325.7 for V2. Like thediversity, the drop in range-weighted richness values evened out by day14, giving average values of 303.0±43.1 for V1 and 299.0±31.6 for V2between days 14 and 28 (p>0.10). We also observed a drop in communityevenness during the initial 10 days of the experiment. The Shannonequitability values dropped slightly from 0.86 to 0.80 for V1 and 0.86to 0.82 for V2. This drop evened out by day 14, giving average values of0.83±0.01 for V1 and 0.82±0.02 for V2 between days 14 and 28 (p>0.10)(Table 6).

To determine the biological significance of our steady-state communitieswe compared the profiles of our vessels immediately after inoculation toour samples from steady-state (day 26), as shown in FIG. 5. We foundthat V1 and V2 were 96.3% similar to each other immediately followinginoculation, and 96.0% similar to each other 26 days post-inoculation.However, V1 day 0 and V1 day 26 were 40.2% similar to each other, whileV2 day 0 and V2 day 26 were 39.3% similar to each other.

TABLE 6 Comparison of two communities cultured separately with fecalinocula from the same healthy donor in Vessel #1 (V1) and Vessel #2(V2). Time period Result of (Days paired Parameter V1 V2 x-y) t-testDynamics (Dy) 25.1 ± 13%  20.8 ± 9.0%  0-10 p > 0.10  1.6 ± 0.2%  3.9 ±1.6% 24-28 p > 0.10 Shannon index 3.03 ± 0.06 2.98 ± 0.10 14-28 p > 0.10(H) Range-weighted 303.0 ± 43.1  299.0 ± 31.6  14-28 p > 0.10 richness(Rr) Shannon 0.83 ± 0.01 0.82 ± 0.02 14-28 p > 0.10 equitability (E_(H))

Example 6. Comparison of Different Media

Our laboratory developed a culture media recipe based on two otherrecipes found in the literature, as described above (Gibson, G. R., etal., Appl. Environ. Microbiol., 54(11): 2750-5, 1988; Lesmes, U. et al.,J. Agric. Food Chem., 56: 5415-5421, 2008). To compare the effectivenessof our culture media (Media 1) we seeded two vessels with the sameinoculum from a healthy donor, but used our media to feed one vessel(V1), while using the media recipe by Walker et al., Appl. Environ.Microbiol. 71 (7):3692-700, 2005 to feed the other vessel (V6).

The inocula used to seed each vessel were very similar to each other andimmediately after inoculation the correlation coefficients of samplestaken from each vessel was 95.5%. Throughout the course of theexperiment, the two vessels varied from each other based on their DGGEprofiles, with correlation coefficient values fluctuating above andbelow the 80% similarity threshold between days 2 and 26 (FIG. 6). Thesevessels were only consistently similar to each other between days 28 and36, with an average correlation coefficient of 88.9±3.1%.

Between days 28 and 36, both vessels had similar low dynamics asdetermined using moving window correlation, with Δt values of 4.4±2.1%for V1 and 5.5±3.7% for V6 (p>0.10). On day 28, when V1 had alreadyreached steady state, V1 and its inoculum shared a correlationcoefficient of 69.4%, while V6 and its inoculum shared a correlationcoefficient of 53.9%. On day 36, when V6 reached steady state, V1 andits inoculum shared a correlation coefficient of 67.3%, while V6 and itsinoculum shared a correlation coefficient of 60.2%.

The diversity of the communities in both vessels was high throughout theduration of the experiment; however an initial drop in diversity wasseen between days 0 and 10. The Shannon-Weaver index values dropped from3.54 to 2.84 for V1 and from 3.52 to 2.63 for V6 during this period.This drop evened out by day 14, giving average values of 3.02±0.06 forV1 and 2.92±0.10 for V6 between days 14 and 36 (p<0.05). The Shannonindex values became similar between days 32 and 36, with average valuesof 2.96±0.01 for V1 and 3.04±0.07 for V6 (p>0.10). Also during theinitial 10 days of this run, the range-weighted richness values droppedin both vessels. These values dropped from 625.2 to 365.5 for V1 andfrom 626.9 to 366.0 for V6. This drop evened out by day 14, givingaverage range-weighted richness values of 333.0±52.7 for V1 and357.8±41.7 for V6 between days 14 and 36 (p>0.10). A drop in communityevenness was also observed between days 0 and 10 of the experiment. TheShannon equitability values dropped from 0.88 to 0.81 for V1 and 0.88 to0.75 for V6. This drop evened out by day 14, giving average values of0.85±0.01 for V1 and 0.83±0.02 for V6 between days 14 and 36 (p<0.05)(Table 7).

TABLE 7 Comparison of communities in two vessels using different media,Vessel #1 (V1; our media) and Vessel #6 (V6; Walker et al., Appl.Environ. Microbiol. 71 (7): 3692-700, 2005 media). Time period Result of(Days paired Parameter V1 V6 x-y) t-test Dynamics (Dy)  4.4 ± 2.1%  5.5± 3.7% 28-36 p > 0.10 Shannon index 3.02 ± 0.06 2.92 ± 0.10 14-36 p <0.05 (H) Range-weighted 333.0 ± 52.7  357.8 ± 41.7  14-36 p > 0.10richness (Rr) Shannon 0.85 ± 0.01 0.83 ± 0.02 14-36 p < 0.05equitability (E_(H))

Example 7. Comparison of Two Different Retention Times

Two vessels modeling the distal gut were run in parallel in an identicalmanner, except that the retention time for V1 was set to 65 hours, whilethe retention time for V2 was set to 24 hours. In this experiment wetested whether an increased retention time would allow the more slowgrowing bacteria to establish themselves within the vessel and thereforethe community, increasing community diversity.

The inocula used to seed each vessel were very similar to each other andimmediately after inoculation the correlation coefficients of samplestaken from each vessel was 96.7%. While V1 and V2 were reaching steadystate they varied from each other and by day 14 the correlationcoefficient between V1 and V2 dropped to 78.0%.

FIG. 7 shows each vessel compared to their respective inocula duringdays 0-10. Over the 10 day period shown V2 was more similar to itsinoculum than V1. V1 had an average correlation coefficient of 48.3±2.9%between days 4 and 10 while V2 had an average correlation coefficient of67.9±5.4% (p<0.01). Comparisons between the inocula and samples taken onday 10 showed that V2 maintained a community that was more similar toits inoculum, with a correlation coefficient of 75.7% for V2 and only50.5% for V1.

Differences in community dynamics were observed during the first 16 daysof the experiment using the two different retention times. Between days10-16, V1 had a Δt value of 4.4±1.9% while V2 had a Δt of 9.9±6.5%(p>0.10). If the experiment had been allowed to run longer, we wouldhave expected to see a decrease in the dynamics of the community to avalue more similar to that of V1, as discussed previously.

An initial drop in community diversity was noted between days 0 and 10of the experiment. During this period the Shannon-Weaver index valuesdropped from 3.44 to 3.06 for V1 and from 3.44 to 3.05 for V2. Thesedrops evened out by day 10 giving average values of 3.11±0.05 for V1 and2.99±0.05 for V2, between days 10 and 16 (p>0.05). There was also aninitial drop in range-weighted richness values for each vessel, withvalues dropping from 636.5 to 614.5 for V1 and from 633.9 to 486.1 forV2 between days 0 and 10. By day 10 V1 and V2 began to have similaraverage range-weighted richness values, with 508.3±78.5 for V1 and433.6±61.2 for V2 between days 10 and 16 (p>0.10). Following the patternobserved in the other experiments, a drop in evenness was observedbetween days 0 and 10. The Shannon equitability values dropped from 0.86to 0.77 for V1 and 0.85 to 0.79 for V2. This drop evened out by day 10,giving average values of 0.80±0.02 for V1 and 0.83±0.01 for V2 betweendays 10 and 16 (p>0.05) (Table 8).

TABLE 8 Comparison of communities in two vessels run with differentretention times, Vessel #1 (V1; retention time of 65 hours) and Vessel#2 (V2; retention time of 24 hours). Time period Result of (Days pairedParameter V1 V2 x-y) t-test Dynamics (Dy)  4.4 ± 1.9% 9.7 ± 5.3 10-16p > 0.10 Shannon index 3.11 ± 0.05 2.99 ± 0.05 10-16 p > 0.05 (H)Range-weighted 508.3 ± 78.5  433.6 ± 61.2  10-16 p > 0.10 richness (Rr)Shannon 0.80 ± 0.02 0.83 ± 0.01 10-16 p > 0.05 equitability (E_(H))

Example 8. Effect of Mucin on Gut Communities

Mucin is an important carbon source for the microbial communities of thedistal colon (Derrien, M. et al., Gut Microbes, 1 (4):254-268, 2010). Todetermine whether mucin addition to our culture media would allow us todevelop communities which are more diverse and more similar to thestarting fecal material, we seeded three vessels with the same inoculumfrom a healthy donor. One vessel was fed using our culture media withoutmucin (V1), while two other vessels were fed using our culture mediasupplemented with 4 g/L mucin (V5 and V6).

The inocula used to seed each vessel were very similar to each other andimmediately after inoculation the similarity of samples taken from eachvessel ranged from 96.1% to 98.1% (FIG. 8). On day 24, the communitiesin V5 and V6 shared a 92.4% similarity, the communities in V1 and V5shared a 61.0% similarity, and the communities in V1 and V6 shared a58.4% similarity (FIG. 8).

The communities in each vessel at day 24 were also compared to thecommunities in each vessel at day 0. For V1, communities at day 0 andday 24 were 56.5% similar; for V5, communities at day 0 and day 24 were59.6% similar; and for V6, communities at day 0 and day 24 were 48.9%similar.

The diversity in each vessel dropped between days 0 and 24, howeverthere was a larger decrease in diversity in the vessel without mucin(V1) than there was in the vessels with mucin (V5, V6), as shown in FIG.8. The average Shannon-Weaver index value of all three vessels on day 0was 3.35±0.02. At 24 days post-inoculation, the Shannon-Weaver indexvalues dropped to 2.96 for V1, 3.08 for V5, and 3.13 for V6.

The richness in each vessel also dropped between days 0 and 24, howeverthere was a slightly larger decrease in richness in the vessel withoutmucin (V1) than there was in the vessels with mucin (V5, V6), as shownin FIG. 8. The average range-weighted richness value of all threevessels on day 0 was 686.8±2.8. At 24 days post-inoculation, therange-weighted richness values dropped to 504.2 for V1, 526.8 for V5,and 526.2 for V6.

Finally, the evenness in each vessel dropped between days 0 and 24.There was a larger decrease in evenness in the vessel without mucin (V1)than there was in the vessels with mucin (V5, V6), as shown in FIG. 8.The average Shannon equitability index value of all three vessels on day0 was 0.84±0.01. At 24 days post-inoculation, the Shannon equitabilityindex values dropped to 0.77 for V1, 0.80 for V5, and 0.81 for V6.

In sum, we have developed and characterized microbial communities fromthe human distal colon that were stable, reproducible, and biologicallysignificant in a single-stage chemostat model of the gut. We fullycharacterized the diversity, stability, and similarity of thesecommunities and also characterized the fecal inoculations from thestarting material to use as a point of reference when analyzing oursimulated communities. We show that the microbial communities arephysiologically relevant, steady-state communities having reproduciblestarting points. The in vitro communities closely mimic the communitiesof the distal gut microbiota. We also compared several fecal inoculafrom Donor 2 over an 8 month period, and found that the predominantbacterial species from this healthy donor remained stable over time (notshown). These values provide us with a baseline to which we can compareour chemostat community values. Our microbial chemostat communitiesmaintained similar high diversity, richness, and evenness values.

The results also show that one can develop reproducible communities inour chemostat model as this donor fulfills our criteria to donate(healthy, no recent history of antibiotics), as one can collect a stoolsample to use in future experiments that will share similar profiles toa stool sample taken at an earlier time. This means that similarsteady-state communities can be established that can be compared betweenchemostat runs using complex microbial communities prepared from freshfecal samples.

Comparison of the fecal community from four different healthy donorsshowed that each donor had a unique profile (as expected, Tannock G. W.,Eur. J. Clin. Nutr., 56 Suppl 4:S44-9, 2002). However, all four profilesshowed similar diversity, richness, and evenness values. This suggeststhat the fecal microbiota from different healthy individuals sharesimilar levels of diversity (as assessed by DGGE).

We used DGGE to assess whether there was a significant differencebetween 10% and 20% inocula used to seed a chemostat vessel in terms ofcommunity structure and diversity. Based on the % similarity, we foundsimilar within- and between-group differences for both concentrations ofinocula. Both communities also had similar community diversity,richness, and evenness values. However, one obvious difference betweenthe two concentrations of inocula is the thickness of the inocula, asthe 20% inoculum was much thicker than the 10% inocula. This madeinoculation with the 20% inocula much more difficult. This, togetherwith the fact that the 10% and 20% inocula were very similar as assessedby DGGE, meant that a 10% inoculum was used for all future studies.

We also compared the two concentrations of inocula to the starting fecalmaterial to assess whether the protocol used to prepare the inoculamight have altered the microbial community structure. We found that boththe 10% and 20% inocula were composed of microbial communities whichwere representative of the starting feces. This result shows that ourprotocol does not cause the inoculum to vary significantly from thefeces it was derived from, making it a relevant seeding material tosimulate the in vivo community in our chemostat model. Differencesbetween the fecal inocula (the supernatant) and the pellet formed aftercentrifugation of the fecal slurry may be due to bacteria adherent tofood residues that did not detach when homogenized. As these populationsprobably represent more specialized niches, they are not representativeof the general luminal populations of interest for our studies.

Example 9. Supplementing Microbial Growth Using Liquid Gold

Liquid Gold was obtained by filter-sterilization of a donor-seededchemostat sample, as described above. In brief, the sample wascentrifuged at 14,000 rpm for 10 minutes and the supernatant wasfiltered sequentially through different sized syringe filters in thefollowing order: 1.00.80.45 μm and finally 0.22 Sequential filtrationwas required to allow removal of sediments, which readily clog thefilters. Liquid Gold was used to supplement FAA plates to a finalconcentration of 3%. Concentrations of 1%, 3%, 5% and 10% have beentested, and 3% was found to be optimal (not shown).

Growth using Liquid Gold-supplemented FAA plates was observed forFaecalibacterium prausnitzii and Ruminococcus callidus (ATCC27760). Forboth of these species, no growth was observed using unsupplemented FAAplates. Liquid Gold-supplemented FAA plates yielded ˜30 colonies forRuminococcus callidus and −50 colonies for F. prausnitzii when streakedfrom frozen stocks.

The F. prausnitzii strain used here was isolated from Donor 5. LiquidGold plates supplemented with Donor 5 Liquid Gold was used to grow thestrain from frozen stock. R. callidus was grown on Donor 5 Liquid Goldsupplemented plates and Donor 6 Liquid Gold supplemented plates. Growthwas observed for both media types, but plates supplemented with Donor 5Liquid Gold yielded more growth (30 colonies versus 5), suggesting thatthere are growth-enhancement relevant differences between Liquid Goldfrom different sources or donors.

FIG. 14 shows that growth of the F. prausnitzii strain isolated fromDonor 5 was enhanced by supplementation of culture media with LiquidGold media supplement from Donor 6. Plates were inoculated withidentical inocula and incubated at 37° C. for 3 days under totalanaerobic conditions. Plate A: Fastidious anaerobe agar supplementedwith 5% defibrinated sheep blood alone; Plate B: Fastidious anaerobeagar supplemented with 5% defibrinated sheep blood and 3% filtered(cell-free) Liquid Gold (from donor 6). FIG. 14 shows that growth wasclearly enhanced by the addition of Liquid Gold to the media at aconcentration of 3%.

We have regularly stored Liquid Gold for several months at 4° C.,without a noticeable diminishment of effectiveness, indicating that thegrowth-enhancing qualities of Liquid Gold are stable.

In summary, the above examples show that the methods described hereinprovide for preparation of stable and reproducible communities, whichcan be used, e.g., to assess the effect of a treatment on communitycomposition and structure. For example, a “test” vessel can be operatedin parallel with a “control” vessel. Running two identical vessels inparallel and ensuring they have identical, steady-state communities atthe time of treatment allows one to determine that shifts in thecommunity are due to the treatment, and not to naturally occurringshifts in the community.

As described above, we monitored the colonization of two identicalvessels set to mimic the distal colon for 28 days post-inoculation. DGGEwas used to monitor the composition, diversity, and dynamics of twoidentical chemostat vessels (V1 and V2) seeded with identical fecalinocula from a healthy donor. We determined that two vessels could berun in parallel and maintain identical communities.

We used moving window correlation to create stability profiles byplotting the % similarity values between day x and day x-2 (FIGS. 5, 7).There was an increase in the rate of change values as the communitiestransitioned from an in vivo to an in vitro environment (days 0-10).During this period there was an initial drop in community diversity inboth vessels. When we looked more closely at the communities byanalyzing the richness and evenness separately, we saw that the drop indiversity was more influenced by the drop in richness than the drop inevenness. After the transition period the communities stabilized and theShannon index, range-weighted richness, and Shannon's equitabilityvalues were identical and reflected a stable community able to maintainhigh diversity, richness, and evenness.

While the two vessels shared a similar community composition by Day 10,they didn't develop identical bacterial communities that were similarboth in terms of species composition and abundance until 26 dayspost-inoculation. Both communities reached steady state as the rate ofchange values dropped below 5% by day 26 (achieved for both vesselsbetween days 24-28).

Taken together, the results reported herein show that our single-stagechemostat vessels can be seeded with the same fecal community andproduce communities that are stable, reproducible, and diverse, reachingsteady state after approximately 26 days post-inoculation. Further, oursingle-stage chemostat was able to develop two identical steady-statecommunities which were more similar to each other than communitiesdeveloped in multi-stage chemostat systems (Van den Abbeele, P. et al.,Appl. Environ. Microbiol., 76(15): 5237-46, 2010). In our single-stagechemostat model of the distal gut we found that the communitiesdeveloped in two identical vessels showed a correlation of 97.6% on day26. At this time the band brightness in these DGGE profiles was almostidentical. Overall, the single-stage chemostat model of the distal gutproduced more stable, reproducible communities than those grownpreviously in multi-stage chemostats.

It is known that the composition of the gut microbiota varies dependingon the segment of the intestine being sampled (Mai, V. and Morris, J. G.Jr., J. Nutr., 134(2):459-64, 2004; Marteau, P. et al., Appl. Environ.Microbiol., 67(10):4939-42, 2001). Fresh fecal samples should be used tomodel the bacterial communities of the distal gut lumen since the fecalbacteria are more representative of the distal gut luminal microbiotathan of the microbiota from other segments of the intestine (Possemiers,S. et al., FEMS Microbiol. Ecol., 49(3): 495-507, 2004). Modelling thedistal gut in a single-stage system more accurately reflects the in vivoenvironment to provide more biologically significant results.

In addition, microbial diversity and community composition within thegut is influenced by several physical, biochemical, and physiologicalfactors. One must assure that the simulated community is as similar tothe in vivo community as possible if the results are to be extrapolatedto the host, so it is important to control and mimic these factors asclosely as possible when designing in vitro simulators.Computer-operated process controls of chemostat models allow forexperimental parameters such as pH, temperature, feed rate, and oxygenlevels to be continuously monitored and automatically adjusted ifdeviations occur.

As described above, to determine the effectiveness of our media recipewe set up two chemostat vessels: one vessel fed with media preparedaccording to our recipe (V1), and another vessel fed with media preparedaccording to a previously published recipe (V6, Walker, et al., Appl.Environ. Microbiol., 71 (7):3692-700, 2005). The community in V1 wasmore similar to its inoculum than the community in V6, meaning that thevessel fed using the Media 1 culture medium supported a community thatwas more representative of the fecal microbiota than the communitysupported by the previously published medium. Both vessels sharedsimilar community dynamics throughout the course of the experiment andsimilar rate of change values between days 28 and 36. Communitydiversity and evenness was higher in V1 between days 14 and 36, however,both vessels shared similar range-weighted richness values during thisperiod. While both media can support diverse communities mimicking thoseof the distal gut, our media recipe supports a community that is moresimilar to the inoculum. Based on these observations, the media recipewe developed provides a suitable medium to grow a stable and diversechemostat community.

As described above, we also investigated whether an increased retentiontime would allow the more slow growing bacteria to establish themselveswithin the vessel during the beginning of the experiment (and thereforeestablish themselves within the community), increasing communitydiversity. We did this to ensure that the system retention timecurrently being used (24 hours) was not high enough to prevent certainslow growing populations from surviving within the system. We set up twochemostat vessels with different retention times (V1 and V2). V1 had alonger retention time (65 hrs), while V2 had a shorter, morebiologically relevant retention time (24 hrs). DGGE analysis showed thatboth vessels developed different communities over time (see Table 8). Ofthese two communities, V2 with a 24 hr retention time developed acommunity that was more similar to its inoculum than the communitydeveloped in V1. Both vessels had similar community dynamics; howeverthe dynamics in V2 was more variable. Both vessels were similar to eachother in terms of community diversity, richness, and evenness. Overall,increasing the retention time from the biologically significant value of24 hours to 65 hours resulted in a community which was less similar toits inoculum and did not maintain a higher level of diversity.

We also compared the colonization process in three chemostat vessels:one vessel fed with a medium without mucin (V1), and two identicalvessels fed with a medium supplemented with mucin (V5 and V6). While nodifferences were observed between all three vessels on day 0, by day 24the vessels supplemented with mucin were similar to each other, butdifferent from the vessel that was not supplemented with mucin. Theaddition of mucin to the chemostat resulted in increases in communitydiversity, richness, and evenness. The addition of mucin also allowedfor the development of communities which were more similar to theinoculum than communities without mucin. These results show that itcould be advantageous, in certain embodiments, to include mucin as partof the culture medium recipe.

Analysis of samples using the Shannon diversity index, range-weightedrichness, and Shannon equitability across several gels resulted invariability in values for the same samples. To correct for this, we ranthe same samples at the end of one gel and the beginning of the nextgel. For example, if the first gel contained the samples from days 0-10,the next gel would contain the samples from days 10-20, and the next gelwould contain the samples from days 20-30, etc. We then added orsubtracted the calculated values for the overlapping days to correct forany variation between gels. While the variability of the Shannondiversity index and Shannon equitability values were relatively small,larger variation was seen in the range-weighted richness values. Whilebetween-gel variability is an inherent drawback of using DGGE, it stillprovides us with estimates of community composition and structure. Moredetailed analyses (such as metagenomic analyses) can also be performed.

In conclusion, we have demonstrated that our single-stage model of thehuman distal gut supported complex communities which were stable,reproducible, and diverse. We also presented data to support ouroptimized operational parameters including inoculum concentration, mediarecipe, and system retention time.

Although this invention is described in detail with reference topreferred embodiments thereof, these embodiments are offered toillustrate but not to limit the invention. It is possible to make otherembodiments that employ the principles of the invention and that fallwithin its spirit and scope as defined by the claims appended hereto.

The contents of all documents and references cited herein are herebyincorporated by reference in their entirety.

We claim:
 1. A method of isolating anaerobic bacteria from human gut,comprising: (a) culturing a fecal sample comprising a microbialcommunity of the human gut in culture media in a single-stage chemostatunder conditions replicating normal human colonic gastrointestinaltract, until equilibrium is reached; (b) diluting the culture andplating onto anaerobe agar supplemented with a media supplement, andoptionally supplemented with defibrinated sheep blood, wherein the mediasupplement comprises a filtrate of effluent from a chemostat vessel inwhich a target bacterial ecosystem has been cultured, wherein the targetbacterial ecosystem has been cultured in media comprising: 0.2% w/vPeptone; 0.2% w/v Yeast extract; 0.2% w/v NaHCO₃; 0.2% w/v Pectin; 0.2%w/v Arabinogalactan; 0.3% w/v Casein; 0.5% w/v unmodified wheat starch;0.1% w/v inulin; 0.05% w/v bile salts; 0.05% w/v L cysteine HCl; 0.0001%w/v CaC1₂; 0.0001% w/v NaCl; 0.0004% w/v K₂HPO₄; 0.0004% w/v KH₂PO₄;0.0001% w/v MgSO₄; 0.00005% w/v Hemin; and 0.00001% w/v menadione; andwherein the target bacterial ecosystem comprises Bacteroides ovatus,Bacteroides vulgatus, Bifidobacterium adolescentis, Bifidobacteriumlongum, Collinsella aerofaciens, Eubacterium rectale, Faecalibacteriumprausnitzii, Parabacteroides distasonis, Roseburia inulinivorans, andRuminococcus obeum; (c) incubating plates in an anaerobe chamber; (d)purifying individual anaerobic bacterial colonies grown in step (c) toisolate anaerobic bacteria from the human gut; and optionally, culturingthe purified individual anaerobic bacterial colonies from step (d) inliquid culture in a single-stage chemostat under conditions replicatingnormal human colonic gastrointestinal tract, optionally wherein themedia supplement of claim 57 is used to supplement culture media atabout 1% v/v to about 10% v/v; such that isolates of anaerobic bacteriaare obtained.
 2. The method of claim 1, wherein the anaerobe chambercontains an atmosphere of N₂, CO₂ or H₂, or a mixture thereof.
 3. Themethod of claim 1, wherein the target bacterial ecosystem comprises ahuman fecal sample.
 4. The method of claim 3, wherein the human fecalsample is a 10% w/v fecal slurry supernatant or a 20% w/v fecal slurrysupernatant.
 5. The method of claim 1, wherein the culture media of stepa) further comprises 0.2% w/v Xylan.
 6. The method of claim 1, whereinthe anaerobic bacteria obtained is Faecalibacterium prausnitzii orRuminococcus callidus (ATCC27760).
 7. The method of claim 1, whereinobtaining at least one of the anaerobic bacteria of Faecalibacteriumprausnitzii, Ruminococcus callidus (ATCC27760), or a Roseburia speciesserves as a positive indicator of effectiveness of the method.
 8. Themethod of claim 1, wherein the fecal sample and the target bacterialecosystem are isolated from the same human fecal sample.